Methods of treating muscular dystrophy

ABSTRACT

Disclosed herein are α7β1 integrin modulatory agents and methods of using such to treat conditions associated with decreased α7β1 integrin expression or activity, including muscular dystrophy. In one example, methods for treating a subject with muscular dystrophy are disclosed. The methods include administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy. Also disclosed are methods of enhancing muscle regeneration, repair, or maintenance in a subject and methods of enhancing α7β1 integrin expression by use of the disclosed α7β1 integrin modulatory agents. Methods of prospectively preventing or reducing muscle injury or damage in a subject are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the U.S. National Stage of International Application No. PCT/US2014/029085, filed Mar. 14, 2014, which was published in English under PCT Article 21(2), which in turn claims the benefit of U.S. Provisional Patent Application No. 61/798,479, filed on Mar. 15, 2013, which is herein incorporated by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under Grant Numbers R43 AR060030, R21 NS058429-01, and R21 AR060769 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

This disclosure relates to the field of muscular dystrophy and in particular, to compositions and methods for treating muscular dystrophy, such as Duchenne muscular dystrophy, Fukuyama congenital muscular dystrophy or merosin deficient congenital muscular dystrophy type 1A or 1D.

BACKGROUND

Mutations in the α7 integrin gene are responsible for congenital myopathy in man. The α7β1 integrin is also a major modifier of muscle disease progression in various genetic muscle diseases including various types of muscular dystrophy, such as Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) and merosin deficient congenital muscular dystrophy type 1A (MDC1A). However, transcriptional regulation of the α7 integrin gene, including such role in muscular dystrophy (e.g., DMD, FCMD and/or MDC1A), remains poorly understood.

Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disease and the most common form of muscular dystrophy. DMD affects 1 in 3500 live male births with patients suffering from chronic muscle degeneration and weakness. Clinical symptoms are first detected between the ages of 2 and 5 years and, by the time the patient is in their teens, the ability for independent ambulation is lost. Death typically occurs in the patient before they are 30 years old due to cardiopulmonary failure.

Fukuyama congenital muscular dystrophy (FCMD) and MDC1A are congenital muscular dystrophies that are heritable neuromuscular disorders. MDC1A is characterized by muscle weakness at birth or in infancy. Affected infants will present with poor muscle tone and few movements. The quality of life and life span of the child is affected through progressive muscle wasting, respiratory compromise, and spinal rigidity. MDC1A is the most common and severe form of congenital muscular dystrophy, accounting for 30-40% of all congenital muscular dystrophy (CMD) diagnosed cases. MDC1A is characterized by congenital hypotonia, distinct joint contractures, and a lack of independent ambulation. Feeding tube placement and positive pressure ventilation is often required for the respiratory problems that occur. Patients afflicted with MDC1A often die before they reach the age of ten years. FCMD is caused by mutations in the fukutin gene, located at human chromosome 9q31. The disease is inherited in an autosomal recessive manner. FCMD is a type of Limb-Girdle muscular dystrophy. Currently there is no cure for DMD, FCMD or MDC1A.

SUMMARY

The muscular dystrophies are a group of diverse, heritable neuromuscular disorders which represent a group of devastating neuromuscular diseases characterized by primary or secondary skeletal muscle involvement. Currently, there are no cures for such diseases.

Disclosed herein are α7β1 integrin expression modulatory agents and methods of using such to treat a condition associated with impaired α7 integrin expression, such as muscular dystrophy. In one embodiment, a method for treating a subject with muscular dystrophy is disclosed. The method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy (such as MDC1A, MDC1D, LGMD, DMD, FCMD or FHMD).

Also disclosed are methods of enhancing muscle regeneration, repair, or maintenance in a subject. In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject in need of muscle regeneration, repair or maintenance, wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject. In a specific embodiment, the present disclosure provides a method for increasing muscle regeneration in a subject. For example, geriatric subjects, subjects suffering from muscle disorders, and subjects suffering from muscle injury, including activity induced muscle injury, such as injury caused by exercise, may benefit from this embodiment.

In yet further embodiments of the disclosed method, the α7β1 integrin modulatory agent is administered in a preventative manner, such as to prevent or reduce muscular damage or injury (such as activity or exercise induced injury). For example, geriatric subjects, subjects prone to muscle damage, or subjects at risk for muscular injury, such as athletes, may be treated in order to eliminate or ameliorate muscular damage, injury, or disease.

Further disclosed are methods of enhancing α7β1 integrin expression. In some embodiments, the method includes contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof and increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression. The methods of the present disclosure can include administering the α7β1 integrin modulatory agent with one or more additional pharmacological substances, such as a therapeutic agent. In some aspects, the additional therapeutic agent enhances the therapeutic effect of the α7β1 integrin modulatory agent. In further aspects, the therapeutic agent provides independent therapeutic benefit for the condition being treated. In various examples, the additional therapeutic agent is a component of the extracellular matrix, such as an integrin, dystrophin, dystroglycan, utrophin, or a growth factor. In further examples, the therapeutic agent reduces or enhances expression of a substance that enhances the formation or maintenance of the extracellular matrix. In some examples, the therapeutic agent is an additional α7β1 integrin modulatory agent such as laminin-111, a laminin-111 fragment, valproic acid or a valproic acid analog.

In some examples, the α7β1 integrin modulatory agent is applied to a particular area of the subject to be treated. For example, the α7β1 integrin modulatory agent may be injected into a particular area to be treated, such as a muscle. In further examples, the α7β1 integrin modulatory agent is administered such that it is distributed to multiple areas of the subject, such as systemic administration or regional administration.

A α7β1 integrin modulatory agent, can be administered by any suitable method, such as topically, parenterally (such as intravenously or intraperitoneally), or orally. In a specific example, the α7β1 integrin modulatory agent is administered systemically, such as through parenteral administration, such as stomach injection or peritoneal injection.

Although the disclosed methods generally have been described with respect to muscle regeneration, the disclosed methods also may be used to enhance repair or maintenance, or prevent damage to, other tissues and organs. For example, the methods of the present disclosure can be used to treat symptoms of muscular dystrophy stemming from effects to cells or tissue other than skeletal muscle, such as impaired or altered brain function, smooth muscles, or cardiac muscles.

The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a digital image illustrating the results of quantitative real-time PCR used to assess Itga7, Itgb1, and Lama2 transcript levels in C2C12 myoblasts and myotubes treated for 24 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), 10 μM MLS001165937-01 (IED-937), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD. * denotes a significant difference in relative transcript levels with **p-value<0.01 and ***p<0.001.

FIG. 2 is a digital image of Western Blots and quantitative analysis of α7 Integrin and GAPDH protein levels in C2C12 myotubes treated for 48 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD. Bands were quantified using Image J software and then graphed as α7 Integrin protein levels relative to GAPDH protein levels. * denotes a significant difference in relative protein levels with **p<0.01.

FIG. 3 is an image of results (fluorescence relative to DMSO at various concentrations of the agent) from a screen using particular embodiments of the disclosed α7β1 integrin modulatory agents.

FIG. 4 is an image of the relative fluorescence generated by various concentrations of beta-galactosidase from the FDG substrate (A). The effects of DMSO concentrations on α7^(+/LacZ) myoblasts (B) and myotube (C) assays were determined to be unacceptably high at concentrations greater than 1%. (D) The α7^(+/LacZ) myotube assay response with varying levels of FDG in the FDG Buffer.

FIG. 5 is an image of graphs illustrating dose-response curves for SU9516 on α7^(+/LacZ) myoblasts (A) or myotubes (B). The non-conventional appearance of the curves is thought to be a mechanism of concentration dependent cellular toxicity.

FIG. 6 is an image of a table providing curve classification results from a primary myoblast screen disclosed herein.

FIG. 7 is an image of graphs of myotube dose-response and the corresponding chemical structures [SU9516 (A), MLS000532969 (B), MLS003126425 (C), MLS001060533 (D), MLS000683232 (E), and MLS000683234 (F)] for each compound examined wherein n=6-9 over multiple days for all points.

FIG. 8 is an image of results obtained for C2C12 myotubes treated with DMSO (n=3) or 12 μM SU9516 (n=3), which were examined for α7 Integrin protein levels by western blotting (A) wherein GapDH protein levels were used as a loading normalization control and the α7 Integrin/GapDH levels were quantitated and graphed for both DMSO and SU9516 treatments (B).

FIG. 9 is an image of results obtained for various different compounds disclosed herein and their effects on α7 integrin levels in human DMD myotubes.

FIG. 10 is an image illustrating chemical structures and names, similarity score relative to Stryka-969, and ITGA7^(+/LacZ) myotube dose-response for 4 compounds with similar structures to Stryka-969.

FIG. 11 is an image of a Western blot analysis for α7 Integrin and α-tubulin, quantitated and graphed wherein significance was determined by unpaired t-test with **p<0.01 and ***p<0.001.

FIG. 12 provides an exemplary synthetic scheme for making specific α7β1 integrin modulatory agents disclosed herein.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Overview of Several Embodiments

Disclosed herein are α7β1 integrin expression modulatory agents and methods of using such to treat a condition associated with impaired α7 integrin expression, such as muscular dystrophy.

In one embodiment, a method for treating a subject with muscular dystrophy is disclosed. The method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy.

In some embodiments, a method for treating a subject with muscular dystrophy, comprises administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent comprises a compound having a formula selected from any one of the following

wherein R¹, R^(1′), R², and R³ each independently is as specified in Table 3, Table 4, Table 5, and/or Table 6. In some embodiments, a combination of any of these compounds, or any other α7β1 integrin modulatory agents disclosed herein may be used. The α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy. In some embodiments, the compound may be selected from any of those provided in any one of Tables 1-16 and 18.

In some embodiments, the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1A (MDC1A), merosin deficient congenital muscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).

In some particular embodiments, the muscular dystrophy is DMD, MDC1A or FCMD.

In one particular embodiment, the muscular dystrophy is DMD.

In some embodiments, the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.

In some embodiments, the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.

In some embodiments, the method further includes selecting a subject with muscular dystrophy.

In some embodiments, the selecting a subject with muscular dystrophy includes diagnosing the subject with muscular dystrophy prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.

In other embodiments, a method of enhancing muscle regeneration, repair, or maintenance in a subject is disclosed.

In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.

In some embodiments, the method includes administering the α7β1 modulatory agent prior to the subject experiencing muscle damage or disease.

In some embodiments, the method is a method of enhancing muscle maintenance in a subject.

In some embodiments, the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.

In some embodiments, the α7β1 integrin modulatory agent is administered to a subject at risk of acquiring a muscle disease or damage, such as an elderly subject.

In some embodiments, the method also includes selecting a subject in need of enhancing muscle regeneration, repair, or maintenance.

In some embodiments, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance includes diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.

In some embodiments, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired production of a component of α7β1 integrin prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.

In some embodiments, the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.

In some embodiments, the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.

In further embodiments, a method of prospectively preventing or reducing muscle injury or damage in a subject is disclosed.

In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby prospectively preventing or reducing muscle injury or damage in the subject.

In some embodiments, the subject is at risk of developing a muscle injury or damage.

In some embodiments, the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.

In some embodiments, the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.

In some embodiments, the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.

In even further embodiments, a method of enhancing α7β1 integrin expression is provided.

In some embodiments, the method includes contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof and increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression.

In some embodiments, the cell is a muscle cell.

In some embodiments, the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.

II. Terms

The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The term “comprises” means “includes.” Thus, “comprising A or B,” means “including A, B, or A and B,” without excluding additional elements.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below.

Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless indicated otherwise, the nomenclature of substituents that are not explicitly defined herein are arrived at by naming the terminal portion of the functionality followed by the adjacent functionality toward the point of attachment.

A person of ordinary skill in the art would recognize that the above definitions are not intended to include impermissible substitution patterns (e.g., methyl substituted with 5 different groups, pentavalent carbon, and the like). Such impermissible substitution patterns are easily recognized by a person of ordinary skill in the art.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. All sequences provided in the disclosed Genbank Accession numbers are incorporated herein by reference as available on Aug. 11, 2011. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of this disclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject one or more agents, such as an agent that increases α7β1 expression and/or treats one or more symptoms associated with muscular dystrophy, by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.

Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, or other molecule of interest. Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject, including treating a subject with a muscular dystrophy).

In some examples, an agent can act directly or indirectly to alter the expression and/or activity of α7β1. In a particular example, a therapeutic agent significantly increases the expression and/or activity of α7β1 (which is a muscular dystrophy associated molecule) thereby treating one or more signs or symptoms associated with muscular dystrophy. An example of a therapeutic agent is one that can increase the expression and/or activity of the α7β1 gene or gene product, for example as measured by a clinical response (such as a decrease in one or more signs or symptoms associated with the muscular dystrophy, an improvement in muscular health, regeneration, repair or maintenance of a muscle cell or tissue). “Improving muscular health” refers to an improvement in muscular health compared with a preexisting state or compared with a state which would occur in the absence of treatment. For example, improving muscular health may include enhancing muscle regeneration, maintenance, or repair. Improving muscular health may also include prospectively treating a subject to prevent or reduce muscular damage or injury. “Regeneration” refers to the repair of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, following injury or damage to at least partially restore the muscle or tissue to a condition similar to which the cells or tissue existed before the injury or damage occurred. Regeneration also refers to facilitating repair of cells or tissue in a subject having a disease affecting such cells or tissue to eliminate or ameliorate the effects of the disease. In more specific examples, regeneration places the cells or tissue in the same condition or an improved physiological condition as before the injury or damage occurred or the condition which would exist in the absence of disease. “Maintenance” of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, refers to maintaining the cells or tissue in at least substantially the same physiological condition, such as maintaining such condition even in the presence of stimulus which would normally cause damage, injury, or disease. “Repair” of cells or tissue, such as muscle cells or tissue (or organs) which includes muscle cells, refers to the physiological process of healing damage to the cells or tissue following damage or other trauma.

A “pharmaceutical agent” is a chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when administered to a subject, alone or in combination with another therapeutic agent(s) or pharmaceutically acceptable carriers. In a particular example, a pharmaceutical agent significantly increases the expression and/or activity of α7β1 thereby treating a condition or disease associated with decreased α7β1 expression/activity, such as muscular dystrophy.

Acyl: H—C(O)—, alkyl-C(O)—, substituted alkyl-C(O)—, cycloalkyl-C(O)—, substituted cycloalkyl-C(O)—, substituted aryl-C(O)—, heteroaryl-C(O)—, substituted heteroaryl-C(O)—, heterocyclyl-C(O)—, and substituted heterocyclyl-C(O)—.

Acylamino: —NR^(a)C(O)alkyl, —NR^(a)C(O)substituted alkyl, —NR^(a)C(O)cycloalkyl, —NR^(a)C(O)substituted cycloalkyl, —NR^(a)C(O)cycloalkenyl, —NR^(a)C(O)substituted cycloalkenyl, —NR^(a)C(O)alkenyl, —NR^(a)C(O)substituted alkenyl, —NR^(a)C(O)alkynyl, —NR^(a)C(O)substituted alkynyl, —NR^(a)C(O)aryl, —NR^(a)C(O)substituted aryl, —NR^(a)C(O)heteroaryl, —NR^(a)C(O)substituted heteroaryl, —NR^(a)C(O)heterocyclyl, and —NR^(a)C(O)substituted heterocyclyl, wherein R^(a) is selected from hydrogen, alkyl, aryl, and cycloalkyl.

Acyloxy: alkyl-C(O)O—, substituted alkyl-C(O)O—, aryl-C(O)O—, substituted aryl-C(O)O—, cycloalkyl-C(O)O—, substituted cycloalkyl-C(O)O—, heteroaryl-C(O)O—, substituted heteroaryl-C(O)O—, heterocyclyl-C(O)O—, and substituted heterocyclyl-C(O)O—.

Acylalkyloxy: alkyl-C(O)alkylO—, substituted alkyl-C(O)alkylO—, aryl-C(O)alkylO—, substituted aryl-C(O)alkylO—, cycloalkyl-C(O)alkylO—, substituted cycloalkyl-C(O)alkylO—, heteroaryl-C(O)alkylO—, substituted heteroaryl-C(O)alkylO—, heterocyclyl-C(O)alkylO—, and substituted heterocyclyl-C(O)alkylO—.

Alkyl: A saturated or unsaturated monovalent hydrocarbon having a number of carbon atoms ranging from one to ten (e.g., C₁₋₁₀alkyl), which is derived from removing one hydrogen atom from one carbon atom of a parent compound (e.g., alkane, alkene, alkyne). An alkyl group may be branched or straight-chain.

Alkenyl: A unsaturated monovalent hydrocarbon having a number of carbon atoms ranging from one to ten (e.g., C₂₋₁₀alkenyl), which has at least one carbon-carbon double bond and is derived from removing one hydrogen atom from one carbon atom of a parent alkene. An alkenyl group may be branched, straight-chain, cyclic, cis, or trans (e.g., E or Z).

Alkynyl: A unsaturated monovalent hydrocarbon having a number of carbon atoms ranging from one to ten (e.g., C₂₋₁₀alkynyl), which has at least one carbon-carbon triple bond and is derived from removing one hydrogen atom from one carbon atom of a parent alkyne. An alkynyl group may be branched, straight-chain, or cyclic.

Alkoxy: —O-alkyl (e.g., methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, sec-butoxy, n-pentoxy).

Alkylthio: —S-alkyl, wherein alkyl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-alkyl, or —S(O)₂-alkyl.

Amino: —NH₂.

Aminocarbonyl: —C(O)N(R^(b))₂, wherein each R^(b) independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both R^(b) are not both hydrogen.

Aminocarbonylalkyl: -alkylC(O)N(R^(b))₂, wherein each R^(b) independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both R^(b) are not both hydrogen.

Aminocarbonylamino: —NR^(a)C(O)N(R^(b))₂, wherein R^(a) and each R^(b) are as defined herein.

Aminodicarbonylamino: —NR^(a)C(O)C(O)N(R^(b))₂, wherein R^(a) and each R^(b) are as defined herein.

Aminocarbonyloxy: —O—C(O)N(R^(b))₂, wherein each R^(b) independently is as defined herein.

Aminosulfonyl: —SO₂N(R^(b))₂, wherein each R^(b) independently is as defined herein.

Analog or Derivative: A compound which is sufficiently homologous to a compound such that it has a similar functional activity for a desired purpose as the original compound. Analogs or derivatives refers to a form of a substance, such as cholestan, which has at least one functional group altered, added, or removed, compared with the parent compound. In some examples, examples of an analog are provided in Tables 1-6, for example. “Functional group” refers to a radical, other than a hydrocarbon radical, that adds a physical or chemical property to a substance.

Aryl: a monovalent aromatic carbocyclic group of from 6 to 15 carbon atoms having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl), which condensed rings may or may not be aromatic provided that the point of attachment is through an atom of the aromatic aryl group.

Aryloxy —O-aryl.

Arylthio —S-aryl, wherein aryl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-aryl, or —S(O)₂-aryl.

Biological activity: The beneficial or adverse effects of an agent on living matter. When the agent is a complex chemical mixture, this activity is exerted by the substance's active ingredient or pharmacophore, but can be modified by the other constituents. Activity is generally dosage-dependent and it is not uncommon to have effects ranging from beneficial to adverse for one substance when going from low to high doses. In one example, the agent significantly increases the biological activity of α7β1 which reduces one or more signs or symptoms associated with the muscular dystrophy.

Contacting: Placement in direct physical association, including both a solid and liquid form. Contacting an agent with a cell can occur in vitro by adding the agent to isolated cells or in vivo by administering the agent to a subject.

Control: A sample or standard used for comparison with a test sample, such as a biological sample obtained from a patient (or plurality of patients) without a particular disease or condition, such as a muscular dystrophy. In some embodiments, the control is a sample obtained from a healthy patient (or plurality of patients) (also referred to herein as a “normal” control), such as a normal biological sample. In some embodiments, the control is a historical control or standard value (e.g., a previously tested control sample or group of samples that represent baseline or normal values (e.g., expression values), such as baseline or normal values of a particular gene such as a α7β1 gene, gene product in a subject without a muscular dystrophy). In some examples, the control is a standard value representing the average value (or average range of values) obtained from a plurality of patient samples (such as an average value or range of values of the gene or gene products, such as the α7β1 gene or gene products, in the subjects without a muscular dystrophy).

Carboxyl: —COOH or salts thereof.

Carboxyester: —C(O)O-alkyl, —C(O)O— substituted alkyl, —C(O)O-aryl, —C(O)O-substituted aryl, —C(O)O-cycloalkyl, —C(O)O-substituted cycloalkyl, —C(O)O-heteroaryl, —C(O)O-substituted heteroaryl, —C(O)O-heterocyclyl, and —C(O)O-substituted heterocyclyl.

(Carboxyester)amino: —NR^(a)—C(O)O-alkyl, —NR^(a)—C(O)O-substituted alkyl, —NR^(a)—C(O)O-aryl, —NR^(a)—C(O)O-substituted aryl, —NR^(a)—C(O)O-cycloalkyl, —NR^(a)—C(O)O-substituted cycloalkyl, —NR^(a)—C(O)O-heteroaryl, —NR^(a)—C(O)O-substituted heteroaryl, —NR^(a)—C(O)O-heterocyclyl, and —NR^(a)—C(O)O-substituted heterocyclyl, wherein R^(a) is as recited herein.

(Carboxyester)oxy: —O—C(O)O-alkyl, —O—C(O)O— substituted alkyl, —O—C(O)O-aryl, O—C(O)O-substituted aryl, —O—C(O)O-cycloalkyl, —O—C(O)O-substituted cycloalkyl, —O—C(O)O-heteroaryl, —O—C(O)O-substituted heteroaryl, —O—C(O)O-heterocyclyl, and —O—C(O)O-substituted heterocyclyl.

Cyano: —CN.

Cycloalkyl: cyclic alkyl (or alkenyl, or alkynyl) groups of from 3 to 10 carbon atoms having single or multiple cyclic rings including fused, bridged, and spiro ring systems (e.g., cyclopropyl, cyclobutyl, etc.).

(Cycloalkyl)oxy: —O-cycloalkyl.

(Cycloalkyl)thio: —S-cycloalkyl. This term also encompasses oxidized forms of sulfur, such as —S(O)— cycloalkyl, or —S(O)₂-cycloalkyl.

Decrease: To reduce the quality, amount, or strength of something. In one example, a therapy decreases one or more symptoms associated with the muscular dystrophy, for example as compared to the response in the absence of the therapy.

Diagnosis: The process of identifying a disease, such as muscular dystrophy, by its signs, symptoms and results of various tests. The conclusion reached through that process is also called “a diagnosis.” Forms of testing commonly performed include blood tests, medical imaging, urinalysis, and biopsy.

Effective amount: An amount of agent that is sufficient to generate a desired response, such as reducing or inhibiting one or more signs or symptoms associated with a condition or disease. When administered to a subject, a dosage will generally be used that will achieve target tissue/cell concentrations. In some examples, an “effective amount” is one that treats one or more symptoms and/or underlying causes of any of a disorder or disease. In some examples, an “effective amount” is a therapeutically effective amount in which the agent alone with an additional therapeutic agent(s) (for example anti-pathogenic agents), induces the desired response such as treatment of a muscular dystrophy, such as DMD, FCMD or MDC1A.

In particular examples, it is an amount of an agent capable of increasing α7β1 gene expression or activity by least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the disease to a point beyond detection).

In some examples, an effective amount is an amount of a pharmaceutical preparation that alone, or together with a pharmaceutically acceptable carrier or one or more additional therapeutic agents, induces the desired response.

In one example, a desired response is to increase the subject's survival time by slowing the progression of the disease, such as slowing the progression of muscular dystrophy. The disease does not need to be completely inhibited for the pharmaceutical preparation to be effective. For example, a pharmaceutical preparation can decrease the progression of the disease by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to the progression typical in the absence of the pharmaceutical preparation.

In another or additional example, it is an amount sufficient to partially or completely alleviate symptoms of the muscular dystrophy within the subject. Treatment can involve only slowing the progression of the disease temporarily, but can also include halting or reversing the progression of the disease permanently.

Effective amounts of the agents described herein can be determined in many different ways, such as assaying for a reduction in of one or more signs or symptoms associated with the muscular dystrophy in the subject or measuring the expression level of one or more molecules known to be associated with the muscular dystrophy. Effective amounts also can be determined through various in vitro, in vivo or in situ assays, including the assays described herein.

The disclosed therapeutic agents can be administered in a single dose, or in several doses, for example daily, during a course of treatment. However, the effective amount can be dependent on the source applied (for example a nucleic acid molecule isolated from a cellular extract versus a chemically synthesized and purified nucleic acid), the subject being treated, the severity and type of the condition being treated, and the manner of administration.

Expression: The process by which the coded information of a gene is converted into an operational, non-operational, or structural part of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For instance, exposure of a cell to a hormone may stimulate expression of a hormone-induced gene. Different types of cells can respond differently to an identical signal. Expression of a gene also can be regulated anywhere in the pathway from DNA to RNA to protein. Regulation can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced. In an example, expression, such as expression of (α7β1, can be regulated to treat one or more signs or symptoms associated with muscular dystrophy.

The expression of a nucleic acid molecule can be altered relative to a normal (wild type) nucleic acid molecule. Alterations in gene expression, such as differential expression, include but are not limited to: (1) overexpression; (2) underexpression; or (3) suppression of expression. Alternations in the expression of a nucleic acid molecule can be associated with, and in fact cause, a change in expression of the corresponding protein.

Protein expression can also be altered in some manner to be different from the expression of the protein in a normal (wild type) situation. This includes but is not necessarily limited to: (1) a mutation in the protein such that one or more of the amino acid residues is different; (2) a short deletion or addition of one or a few (such as no more than 10-20) amino acid residues to the sequence of the protein; (3) a longer deletion or addition of amino acid residues (such as at least 20 residues), such that an entire protein domain or sub-domain is removed or added; (4) expression of an increased amount of the protein compared to a control or standard amount; (5) expression of a decreased amount of the protein compared to a control or standard amount; (6) alteration of the subcellular localization or targeting of the protein; (7) alteration of the temporally regulated expression of the protein (such that the protein is expressed when it normally would not be, or alternatively is not expressed when it normally would be); (8) alteration in stability of a protein through increased longevity in the time that the protein remains localized in a cell; and (9) alteration of the localized (such as organ or tissue specific or subcellular localization) expression of the protein (such that the protein is not expressed where it would normally be expressed or is expressed where it normally would not be expressed), each compared to a control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1A) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.

Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.

Extracellular matrix: An extracellular structure of a tissue or a layer thereof, including the arrangement, composition, and forms of one or more matrix components, such as proteins, including structural proteins such as collagen and elastin, proteins such as fibronectin and laminins, and proteoglycans. The matrix may comprise fibrillic collagen, having a network of fibers. In some examples, the extracellular matrix is connected to cells through the costameric protein network.

Halogen or Halo: fluoro, chloro, bromo, and iodo.

Heteroaryl: an aromatic group of from 1 to 10 carbon atoms and 1 to 4 heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridinyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl), wherein the condensed rings may or may not be aromatic and/or contain a heteroatom, provided that the point of attachment is through an atom of the aromatic heteroaryl group. In one embodiment, the nitrogen and/or sulfur ring atom(s) of the heteroaryl group are optionally oxidized to provide for the N-oxide (N→O), sulfinyl, or sulfonyl moieties.

Heteroaryloxy: —O-heteroaryl.

Heteroarylthio: —S-heteroaryl. This term also encompasses oxidized forms of sulfur, such as —S(O)— heteroaryl, or —S(O)₂-heteoaryl.

Heterocyclyl: a saturated, unsaturated group, or combinations thereof, having a single ring or multiple condensed rings, including fused bridged and spiro ring systems, and having from 3 to 15 ring atoms, including 1 to 4 heteroatoms, selected from nitrogen, sulfur, or oxygen. These groups may be substituted with one or more of the substituents disclosed herein for substituted aryl and/or substituted alkyl. These groups encompass, for example, a saturated heterocyclyl fused with one or more aromatic hydrocarbons or heteroaryl groups.

Heterocyclyloxy: —O-heterocycyl.

Heterocyclylthio: —S-heterocycyl. This term also encompasses oxidized forms of sulfur, such as —S(O)-heterocyclyl, or —S(O)₂-heterocyclyl.

Hydroxyl or Hydroxy: —OH.

Imino: —N═R^(c) wherein R^(c) may be selected from hydrogen, aminocarbonylalkyloxy, substituted aminocarbonylalkyloxy, aminocarbonylalkylamino, and substituted aminocarbonylalkylamino.

Increase: To enhance the quality, amount, or strength of something. In one example, an agent increases the activity or expression of α7β1, for example relative to an absence of the agent. In a particular example, an agent increases the activity or expression of α7β1 by at least 10%, at least 20%, at least 50%, or even at least 90%, including between 10% to 95%, 20% to 80%, 30% to 70%, 40% to 50%, such as 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100%. Such increases can be measured using the methods disclosed herein.

In a particular example, a therapy increases (also known as up-regulates) the expression of α7β1, such as an increase of at least 10%, at least 20%, at least 50%, or even at least 90% in α7β1 expression, thereby treating/alleviating one or more signs or symptoms associated with muscular dystrophy. In some examples, an increase in expression refers to an increase in α7β1 gene product. An α7β1 gene product can be RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein.

Gene upregulation includes any detectable increase in the production of a α7β1 gene product. In certain examples, production of α7β1 gene product increases by at least 2-fold, for example at least 3-fold or at least 4-fold, as compared to a control (such an amount of gene expression in a normal cell). In one example, a control is a relative amount of α7 gene expression or protein expression in a biological sample taken from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1A. Such increases can be measured using the methods disclosed herein. For example, “detecting or measuring expression of α7β1” includes quantifying the amount of the gene, gene product or modulator thereof present in a sample. Quantification can be either numerical or relative. Detecting expression of the gene, gene product or modulators thereof can be achieved using any method known in the art or described herein, such as by measuring nucleic acids by PCR (such as RT-PCR) and proteins by ELISA. In primary embodiments, the change detected is an increase or decrease in expression as compared to a control, such as a reference value or a healthy control subject. In some examples, the detected increase or decrease is an increase or decrease of at least two-fold compared with the control or standard. Controls or standards for comparison to a sample, for the determination of differential expression, include samples believed to be normal (in that they are not altered for the desired characteristic, for example a sample from a subject who does not have muscular dystrophy, such as DMD, FCMD or MDC1A) as well as laboratory values (e.g., range of values), even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory.

Laboratory standards and values can be set based on a known or determined population value and can be supplied in the format of a graph or table that permits comparison of measured, experimentally determined values.

In other embodiments of the methods, the increase or decrease is of a diagnostically significant amount, which refers to a change of a sufficient magnitude to provide a statistical probability of the diagnosis.

The level of expression in either a qualitative or quantitative manner can detect nucleic acid or protein. Exemplary methods include microarray analysis, RT-PCR, Northern blot, Western blot, and mass spectrometry.

Inhibiting a disease or condition: A phrase referring to reducing the development of a disease or condition, for example, in a subject who is at risk for a disease or who has a particular disease. Particular methods of the present disclosure provide methods for inhibiting muscular dystrophy.”

Integrin: A cell surface transmembrane glycoprotein receptor. Integrins are involved in many biological processes such as wound healing, blood clot formation, gene regulation, and immune responses. Integrins can regulate tissue specific cell adhesion molecules. Integrins are heterodimeric non-covalently associated glycoproteins composed of two subunits. The subunits, which are designated α and beta, have approximate molecular weights of 150-180 kilodaltons and 90-110 kilodaltons, respectively.

The α7β1 integrin is a major laminin receptor expressed in skeletal muscle. The α7β1 integrin plays a role in the development of neuromuscular and myotendinous junctions. In the adult, the α7β1 integrin is concentrated at junctional sites and found in extrajunctional regions where it mediates the adhesion of the muscle fibers to the extracellular matrix. Mice that lack the α7 chain develop muscular dystrophy that affects the myotendinous junctions. The absence of α7 integrin results in defective matrix deposition at the myotendinous junction. Loss of the α7 integrin in γ-sarcoglycan mice results in severe muscle pathology. Absence of the α7 integrin in mdx mice also results in severe muscular dystrophy, confirming that the α7β1 integrin serves as a major genetic modifier for Duchenne and other muscular dystrophies.

Mutations in the α7 gene are responsible for muscular dystrophy in humans. A screen of 117 muscle biopsies from patients with undefined muscle disease revealed 3 which lacked the α7 integrin chain and had reduced levels of β1D integrin chain. These patients exhibited delayed developmental milestones and impaired mobility consistent with the role for the α7β1 integrin in neuromuscular and myotendinous junction development and function.

Several lines of evidence suggest the α7 integrin may be important for muscle regeneration. For example, during embryonic development, the α7β1 integrin regulates myoblast migration to regions of myofiber formation. It has been found that MyoD (myogenic determination protein) transactivates α7 integrin gene expression in vitro, which would increase α7 integrin levels in activated satellite cells. Human, mouse and rat myoblast cell lines derived from satellite cells express high levels of α7 integrin. Elevated α7 integrin mRNA and protein are detected in the skeletal muscle of 5 week old mdx mice, which correlates with the period of maximum muscle degeneration and regeneration. In addition, the α7β1 integrin associates with muscle specific β1-integrin binding protein (MIBP), which regulates laminin deposition in C2C12 myoblasts. Laminin provides an environment that supports myoblast migration and proliferation. Finally, enhanced expression of the α7 integrin in dystrophic skeletal muscle results in increased numbers of satellite cells.

The sequences for α7β1 integrin subunits are publicly available on GenBank, see, for example Gene Accession No. NM_001144116 (human) and NM_008398.2 (mouse) for α7 integrin, and Gene Accession No. NM_002211 for β1 integrin (also known as CD29), each of which is herein incorporated by reference as available on Sep. 8, 2011. Exemplary α7β1 integrin modulatory agents are disclosed herein, such as a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.

A α7β1 integrin associated condition is a condition associated with altered α7β1 integrin expression or activity, including muscular dystrophy, such as DMD, FCMD, LGMD, FHMD, Beckers muscular dystrophy and/or MDC1A.

Laminin: Any of the family of glycoproteins that are typically involved in the formation and maintenance of extracellular matrices. Laminin is a heterotrimers formed from an α chain, a β chain, and a γ chain. The various chains of a particular laminin can affect the properties of the molecule. In some aspects of the present disclosure, fragments, derivatives, or analogs of various laminins can be used, such as laminins having at least a portion at least substantially homologous to the laminin α1 chain. A “fragment of laminin,” as used herein, refers to a portion of a substance, such as laminin. A fragment may be, in some examples, a particular domain or chain of a protein. For example, particular embodiments of the present disclosure involve administering a fragment of laminin-1 corresponding to at least a portion of (or all of) the laminin α1 chain. Fragments may be synthetic or may be derived from larger parent substances.

In some aspects, laminins may be administered as a mixture of laminins, including fragments, analogs, and derivatives thereof. Suitable methods for preparing analogs of laminin domains are disclosed in U.S. Pat. No. 6,933,280, incorporated by reference herein to the extent not inconsistent with this disclosure.

The laminin materials or compositions of the present disclosure may be delivered as discrete molecules or may be complexed with, or conjugated to, another substance. For example, the laminin may be combined with a carrier, such as to aid in delivery of the laminin to a site of interest or to increase physiological uptake or incorporation of the laminin.

In specific examples, the laminin administered includes or consists of laminin-1 (LAM-111), which includes the chains α1β1γ1. In further examples, the laminin administered includes or consists of laminin-2, which includes the chains α2β1γ1. In yet further examples, the laminin administered includes or consists of laminin-4, which includes the chains α2β2γ1.

Laminins may be obtained from any suitable source. For example, laminin-1 may be obtained from placental tissue or from Engelbreth-Holm-Swarm murine sarcoma. Suitable methods of isolating various laminins are disclosed in U.S. Pat. No. 5,444,158, incorporated by reference herein to the extent not inconsistent with the present disclosure.

Muscle: Any myoblast, myocyte, myofiber, myotube or other structure composed of muscle cells. Muscles or myocytes can be skeletal, smooth, or cardiac. Muscle may also refer to, in particular implementations of the present disclosure, cells or other materials capable of forming myocytes, such as stem cells and satellite cells.

Muscular dystrophy: A term used to refer to a group of genetic disorders that lead to progressive muscle weakness. Muscular dystrophy can result in skeletal muscle weakness and defects in skeletal muscle proteins, leading to a variety of impaired physiological functions. No satisfactory treatment of muscular dystrophy exists. Existing treatments typically focus on ameliorating the effects of the disease and improving the patient's quality of life, such as through physical therapy or through the provision of orthopedic devices.

Mutated genes associated with muscular dystrophy are responsible for encoding a number of proteins associated with the costameric protein network. Such proteins include laminin-2, collagen, dystroglycan, integrins, caveolin-3, ankyrin, dystrophin, α-dystrobrevin, vinculin, plectin, BPAG1b, muscle LIM protein, desmin, actinin-associated LIM protein, α-actin, titin, telethonin, cypher, myotilin, and the sarcoglycan/sarcospan complex.

The most common form of muscular dystrophy is DMD, affecting 1 in 3,500 live male births. DMD is an X-linked recessive disorder characterized by a mutation in the gene that codes for dystrophin. Dystrophin is a cytoskeletal protein about 430 kDa in size. This protein works to connect the cell's cytoskeleton and extracellular matrix. The loss of dystrophin in DMD patients leads to a loss of muscle fiber attachment at the extracellular matrix during contraction, which ultimately leads to progressive fiber damage, membrane leakage and a loss of muscle function. Most patients die before they reach the age of 30 due to respiratory or cardiac failure.

Beckers muscular dystrophy (also known as Benign pseudohypertrophic muscular dystrophy) is related to DMD in that both result from a mutation in the dystrophin gene, but in DMD no functional dystrophin is produced making DMD much more severe than BMD. BMD is an X-linked recessive inherited disorder characterized by slowly progressive muscle weakness of the legs and pelvis. BMD is a type of dystrophinopathy, which includes a spectrum of muscle diseases in which there is insufficient dystrophin produced in the muscle cells, results in instability in the structure of muscle cell membrane. This is caused by mutations in the dystrophin gene, which encodes the protein dystrophin. The pattern of symptom development of BMD is similar to DMD, but with a later, and much slower rate of progression.

Congenital muscular dystrophies are caused by gene mutations. FCMD and MDC1A are examples of congenital muscular dystrophies. MDC1A is a congenital muscular dystrophy due to a genetic mutation in the LAMA2 gene which results in lack of or complete loss of laminin-α2 protein. This loss of laminin-α2 leads to an absence of laminins-211/221. Laminins-211/221 are major components of the extracellular matrix and play a key role in muscle cell development. During muscle cell differentiation laminin binds to the α7β1 integrin. Without laminin-α2, muscle fibers are unable to adhere to the basement membrane and myotubes undergo apotosis. Muscle regeneration also fails, leading to a loss of muscle repair and an increase in muscle fibrosis and inflammation. This chronic tissue injury is a major cause of morbidity and mortality in MDC1A.

Congenital Muscular Dystrophies (CMD) and Limb-Girdle muscular dystrophy (LGMD) are common forms of highly heterogeneous muscular dystrophies which can be distinguished by their age at onset. In CMD, onset of symptoms is at birth or within the first 6 months of life; in LGMD onset of symptoms is in late childhood, adolescence or even adult life. Inheritance in LGMD can be autosomal dominant (LGMD type 1) or autosomal recessive (LGMD type 2), CMD is recessively inherited. CMD and LGMD can overlap both clinically and genetically

MDC1A is a progressive muscle wasting disease that results in children being confined to a wheelchair, requiring ventilator assistance to breathe and premature death. Symptoms are detected at birth with poor muscle tone and “floppy” baby syndrome. DMD, BMD and LGMD are progressive muscle degenerative diseases usually diagnosed at 3-5 years of age when children show developmental delay including ability to walk and climb stairs. The disease is progressive and children are usually confined to a wheelchair in their teens and require ventilator assistance.

Fukuyama congenital muscular dystrophy (FCMD) is an inherited condition that predominantly affects the muscles, brain, and eyes. Congenital muscular dystrophies are a group of genetic conditions that cause muscle weakness and wasting (atrophy) beginning very early in life. Fukuyama congenital muscular dystrophy affects the skeletal muscles, which are muscles the body uses for movement. The first signs of the disorder appear in early infancy and include a weak cry, poor feeding, and weak muscle tone (hypotonia). Weakness of the facial muscles often leads to a distinctive facial appearance including droopy eyelids (ptosis) and an open mouth. In childhood, muscle weakness and joint deformities (contractures) restrict movement and interfere with the development of motor skills such as sitting, standing, and walking. Fukuyama congenital muscular dystrophy also impairs brain development. People with this condition have a brain abnormality called cobblestone lissencephaly, in which the surface of the brain develops a bumpy, irregular appearance (like that of cobblestones). These changes in the structure of the brain lead to significantly delayed development of speech and motor skills and moderate to severe intellectual disability. Social skills are less severely impaired. Most children with Fukuyama congenital muscular dystrophy are never able to stand or walk, although some can sit without support and slide across the floor in a seated position. More than half of all affected children also experience seizures. Other signs and symptoms of Fukuyama congenital muscular dystrophy include impaired vision, other eye abnormalities, and slowly progressive heart problems after age 10. As the disease progresses, affected people may develop swallowing difficulties that can lead to a bacterial lung infection called aspiration pneumonia. Because of the serious medical problems associated with Fukuyama congenital muscular dystrophy, most people with the disorder live only into late childhood or adolescence.

Fukuyama congenital muscular dystrophy is seen almost exclusively in Japan, where it is the second most common form of childhood muscular dystrophy (after Duchenne muscular dystrophy). Fukuyama congenital muscular dystrophy has an estimated incidence of 2 to 4 per 100,000 Japanese infants.

Fukuyama congenital muscular dystrophy is caused by mutations in the FKTN gene which encodes fukutin. The most common mutation in the FKTN gene reduces the amount of fukutin produced within cells. A shortage of fukutin likely prevents the normal modification of α-dystroglycan, which disrupts that protein's normal function. Without functional α-dystroglycan to stabilize muscle cells, muscle fibers become damaged as they repeatedly contract and relax with use. The damaged fibers weaken and die over time, leading to progressive weakness and atrophy of the skeletal muscles.

Defective α-dystroglycan also affects the migration of neurons during the early development of the brain. Instead of stopping when they reach their intended destinations, some neurons migrate past the surface of the brain into the fluid-filled space that surrounds it. Because Fukuyama congenital muscular dystrophy involves a malfunction of α-dystroglycan, this condition is described as a dystroglycanopathy.

Facioscapulohumeral muscular dystrophy (FHMD) is a form of muscular dystrophy associated with progressive muscle weakness and loss of muscle tissue. Unlike DMD and BMD which mainly affect the lower body, FSHD affects the upper body mainly the face, shoulder and upper arm muscles. However, it can affect muscles around the pelvis, hips, and lower leg. Symptoms for FSHD often do not appear until age 10-26, but it is not uncommon for symptoms to appear much later. In some cases, symptoms never develop. Symptoms are usually mild and very slowly become worse. Facial muscle weakness is common, and may include eyelid drooping, inability to whistle, decreased facial expression, depressed or angry facial expression, difficulty pronouncing words, shoulder muscle weakness (leading to deformities such as pronounced shoulder blades (scapular winging) and sloping shoulders), weakness of the lower, hearing loss and possible heart conditions.

Oxo: (═O).

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more α7β1 modulatory agents.

In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.

Sample (or biological sample): A biological specimen containing genomic DNA, RNA (including mRNA), protein, or combinations thereof, obtained from a subject. Examples include, but are not limited to, peripheral blood, urine, saliva, tissue biopsy, surgical specimen, and autopsy material. In one example, a sample includes muscle biopsy, such as from a subject with DMD, FCMD, or MDC1A.

Signs or symptoms: Any subjective evidence of disease or of a subject's condition, e.g., such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state. A “sign” is any abnormality indicative of disease, discoverable on examination or assessment of a subject. A sign is generally an objective indication of disease. Signs include, but are not limited to any measurable parameters such as tests for detecting muscular dystrophy, including measuring creatine kinase levels, electromyography (to determine if weakness is caused by destruction of muscle tissue rather than by damage to nerves) or immunohistochemistry/immunoblotting/immunoassay (e.g., ELISA) to measure muscular dystrophy-associated molecules, such as α7β1 integrin. In one example, reducing or inhibiting one or more symptoms or signs associated with muscular dystrophy, includes increasing the activity or expression of α7β1 integrin by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to the activity and/or expression in the absence of the treatment. Symptoms of muscular dystrophy include, but are not limited to, muscle weakness and loss, difficulty running, difficulty hopping, difficulty jumping, difficulty walking, difficulty breathing, fatigue, skeletal deformities, muscle deformities (contractions of heels; pseudohypertrophy of calf muscles), heart disease (such as dilated cardiomyopathy), elevated creatine phosphokinase (CK) levels in blood or combinations thereof.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and non-human mammals.

Substituted Alkyl: an alkyl (or alkenyl, or alkynyl) group having from 1 to 5 hydrogen atoms replaced with substituents selected alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, acylalkyloxy, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aminodicarbonylamino, aminocarbonylalkyl, aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, aminodiacylamino, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, imino, oxo, sulfonylamino, nitro, SO₃H, sulfonyl, thiol, imino, substituted imino, alkylthio, and substituted alkylthio. The alkyl may be substituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which are defined herein.

Substituted Alkoxy: —O-(substituted alkyl).

Substituted Alkylthio: —S-(substituted alkyl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted alkyl, or —S(O)₂-substituted alkyl.

Substituted Amino: —N(R^(b))₂, wherein each R^(b) independently is selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl, substituted cycloalkyl, heteroaryl, substituted heteroaryl, heterocyclyl, substituted heterocyclyl. Also, each R^(b) may optionally be joined together with the nitrogen bound thereto to form a heterocyclyl or substituted heterocyclyl group, provided that both R^(b) are not both hydrogen.

Substituted Aryl: aryl groups having 1 to 5 hydrogens replaced with substituents independently selected from alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO₃H, sulfonyl, thiol, alkylthio, and substituted alkylthio. The aryl group may be substituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which are defined herein.

Substituted Aryloxy: —O-(substituted aryl).

Substituted Arylthio: —S-(substituted aryl), wherein substituted aryl is as defined herein. This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted aryl, or —S(O)₂-substituted aryl.

Substituted Cycloalkyl: cycloalkyl, cycloalkenyl, or cycloalkynyl group having from 1 to 5 substituents selected from the group consisting of oxo, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminocarbonylamino, aminocarbonyloxy, aminosulfonyl, aryl, substituted aryl, aryloxy, substituted aryloxy, arylthio, substituted arylthio, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, cycloalkyl, substituted cycloalkyl, cycloalkyloxy, substituted cycloalkyloxy, cycloalkylthio, substituted cycloalkylthio, halo, hydroxy, heteroaryl, substituted heteroaryl, heteroaryloxy, substituted heteroaryloxy, heteroarylthio, substituted heteroarylthio, heterocyclyl, substituted heterocyclyl, heterocyclyloxy, substituted heterocyclyloxy, heterocyclylthio, substituted heterocyclylthio, nitro, SO₃H, sulfonyl, thiol, alkylthio, and substituted alkylthio. The aryl group may be substituted with 1 to 2, 1 to 3, or 1 to 4 of these groups, which are defined herein. In some embodiments, the cycloalkyl group may have multiple condensed rings (e.g. tetrahydronaphthyl or tetrahydroanthacenyl), provided that the point of attachment is through an atom of the nonaromatic ring.

Substituted (Cycloalkyl)oxy: —O-(substituted cycloalkyl).

Substituted (Cycloalkyl)thio: refers to —S-(substituted cycloalkyl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted cycloalkyl, or —S(O)₂-substituted cycloalkyl.

Substituted Heteroaryl: heteroaryl groups that are substituted with from 1 to 5 substituents selected from the group consisting of the same group of substituents defined for substituted aryl.

Substituted Heteroaryloxy: —O-(substituted heteroaryl).

Substituted Heteroarylthio: —S-(substituted heteroaryl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted heteroaryl, or —S(O)₂-substituted heteoaryl.

Substituted Heterocycyloxy: —O-(substituted heterocyclyl) wherein the heterocyclyl group is substituted with one or more of the substituents recited for substituted alkyl.

Substituted Heterocycylthio: —S-(substituted heterocycyl). This term also encompasses oxidized forms of sulfur, such as —S(O)-substituted heterocyclyl, or —S(O)₂-substituted heterocyclyl.

Sulfonyl: —SO₂-alkyl, —SO₂-substituted alkyl, —SO₂-cycloalkyl, —SO₂-substituted cycloalkyl, —SO₂-aryl, —SO₂-substituted aryl, —SO₂-heteroaryl, —SO₂-substituted heteroaryl, —SO₂-heterocyclyl, and —SO₂-substituted heterocyclyl.

Sulfonylamino: —NRaSO₂alkyl, —NRaSO₂substituted alkyl, —NRaSO₂cycloalkyl, —NRaSO₂substituted cycloalkyl, —NRaSO₂aryl, —NRaSO₂substituted aryl, —NR^(a)SO₂heteroaryl, —NRaSO₂substituted heteroaryl, —NR^(a)SO₂heterocyclyl, —NRaSO₂substituted heterocyclyl, wherein each R^(a) independently is as defined herein.

Thiol: —SH.

Thiocarbonyl: (═S)

Tissue: An aggregate of cells, usually of a particular kind, together with their intercellular substance that form one of the structural materials of an animal and that in animals include connective tissue, epithelium, muscle tissue, and nerve tissue.

Treating a disease: A therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to a muscular dystrophy, such as a sign or symptom of muscular dystrophy. Treatment can induce remission or cure of a condition or slow progression, for example, in some instances can include inhibiting the full development of a disease, for example preventing development of a muscular dystrophy. Prevention of a disease does not require a total absence of disease. For example, a decrease of at least 50% can be sufficient.

Treating a disease can be a reduction in severity of some or all clinical symptoms of the disease or condition, a reduction in the number of relapses of the disease or condition, an improvement in the overall health or well-being of the subject, by other parameters well known in the art that are specific to the particular disease or condition, and combinations of such factors.

Under conditions sufficient for: A phrase that is used to describe any environment that permits the desired activity. In one example, includes administering a disclosed agent to a subject sufficient to allow the desired activity. In particular examples, the desired activity is increasing the expression or activity of α7β1.

III. Compounds for Treating Muscular Dystrophy

Disclosed herein are compounds that may be used as α1β7 integrin modulatory agents in methods disclosed herein. In particular disclosed embodiments, the compound is effective in treating muscular dystrophy. The compound is a small-molecule therapeutic. In particular disclosed embodiments, the small-molecule therapeutic is a cyclic compound comprising a heteroatom-containing skeleton. In other disclosed embodiments, the small-molecule therapeutic is a cyclic compound comprising an all-carbon skeleton. In certain disclosed embodiments, the cyclic compound comprising a heteroatom-containing skeleton has a formula illustrated below:

wherein each R¹ independently is selected from C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acylC₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonylC₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, and substituted C₁₋₁₀alkythio, thiocarbonyl; or

two R¹ substituents, together with the atom to which each is bound, may form ring selected from a C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₂₋₁₀substituted heterocyclyl, and C₂₋₁₀heterocyclyloxy, substituted;

each of A, B, C, D, and E independently may be selected from carbon, nitrogen, oxygen, and sulfur; and

n may be zero, 1, 2, 3, 4, or 5.

In other embodiments, the cyclic compound comprising a heteroatom-containing moiety has a formula illustrated below:

wherein each R² independently is selected from C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, C₁₋₁₀alkoxy, substituted C₁₋₁₀alkoxy, acyl, acylamino, acyloxy, acylC₁₋₁₀alkyloxy, amino, substituted amino, aminoacyl, aminocarbonylC₁₋₁₀alkyl, aminocarbonylamino, aminodicarbonylamino, aminocarbonyloxy, aminosulfonyl, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₆₋₁₅aryloxy, substituted C₆₋₁₅aryloxy, C₆₋₁₅arylthio, substituted C₆₋₁₅arylthio, carboxyl, carboxyester, (carboxyester)amino, (carboxyester)oxy, cyano, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, (C₃₋₈cycloalkyl)oxy, substituted (C₃₋₈cycloalkyl)oxy, (C₃₋₈cycloalkyl)thio, substituted (C₃₋₈cycloalkyl)thio, halo, hydroxyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₁₋₁₀heteroaryloxy, substituted C₁₋₁₀heteroaryloxy, C₁₋₁₀heteroarylthio, substituted C₁₋₁₀heteroarylthio, C₂₋₁₀heterocyclyl, C₂₋₁₀substituted heterocyclyl, C₂₋₁₀heterocyclyloxy, substituted C₂₋₁₀heterocyclyloxy, C₂₋₁₀heterocyclylthio, substituted C₂₋₁₀heterocyclylthio, imino, oxo, sulfonyl, sulfonylamino, thiol, C₁₋₁₀alkylthio, and substituted C₁₋₁₀alkythio, thiocarbonyl; or

two R² substituents, together with the atom to which each is bound, may form ring selected from a C₆₋₁₅aryl, substituted C₆₋₁₅aryl, C₃₋₈cycloalkyl, substituted C₃₋₈cycloalkyl, C₁₋₁₀heteroaryl, substituted C₁₋₁₀heteroaryl, C₂₋₁₀substituted heterocyclyl, and C₂₋₁₀heterocyclyloxy, substituted;

each of A, B, C, D, E, and F independently may be selected from carbon, nitrogen, oxygen, and sulfur; and

n may be zero, 1, 2, 3, 4, or 5.

In particular disclosed embodiments, the cyclic compound comprising an all-carbon skeleton may have a general formula provided below:

wherein R³ and R⁴ independently may be selected from hydroxyl, hydrogen, C₁₋₁₀alkyl, substituted C₁₋₁₀alkyl, carboxyl, acyl, aminoacyl, acylamino, amino, substituted amino, C₆₋₁₅aryl, substituted C₆₋₁₅aryl, and C₁₋₁₀alkoxy; R⁵ is selected from amino, substituted amino, oxo, hydroxyl, C₁₋₁₀alkoxy, and imino; and n may be zero, 1, 2, 3, 4, or 5. A person of ordinary skill in the art will recognize that the dashed lines indicate optional bonds which may be present in certain compounds and not present in others.

In particular disclosed embodiments, rings A and B are connected via the optional bonds to form a steroid-based skeleton. In embodiments wherein rings A and B are connected, R⁵ may be bound to ring A via a double bond or a single bond, a feature that is indicated with the optional dashed bond in Formula 13. For example, if R⁵ is amino, hydroxyl, substituted amino, or C₁₋₁₀alkoxy, then R⁵ is attached to ring A via a single bond, whereas if R⁵ is oxo or imino, then R₅ is attached to ring A via a double bond.

In particular disclosed embodiments, C₆₋₁₅aryl may be selected from phenyl, biphenyl, naphthalene, anthracene, and the like; substituted C₆₋₁₅aryl may be selected from phenyl, biphenyl, naphthalene, and anthracene substituted with one or more substituents as defined herein; C₁₋₁₀alkyl may be selected from C₁₋₁₀alkane, C₂₋₁₀alkene, and C₂₋₁₀alkyne; more typically from methyl, ethyl, propyl, butyl, pentyl, hexyl, and the like; ethylene, propylene, butylene, and the like; and ethyne, propyne, butyne, and the like; substituted C₁₋₁₀alkyl may be selected from C₁₋₁₀alkane, C₂₋₁₀alkene, and C₂₋₁₀alkyne substituted with one or of the substituents as provided herein.

Exemplary embodiments concerning hetercyclyl and heteroaryl substitutents include, but are not limited to, epoxy, pyrrolyl, imidazole, pyrazole, pyridinyl, pyrazine, pyrimidine, oxanyl, thianyl, dioxanyl, dithianyl, coumarin, pyridazine, indolizine, isoindole, indolyl, indolinyl (or dihydroindole), indazole, purine, isoquinoline, quinoline, benzo[d]pyridazine, naphthyridine, quinoxaline, quinazoline, benzopyridazine, pteridine, carbazole, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, indoline, phthalimide, 1,2,3,4-tetrahydroisoquinoline, 4,5,6,7-tetrahydrobenzo[b]thiophene, thiazole, thiazolidine, thiophene, benzo[b]thiophene, morpholinyl, thiomorpholinyl, piperidinyl, pyrrolidinyl, piperazinyl, oxazolidinyl, oxazolyl, thiophenyl, isooxazolidinyl, and tetrahydrofuranyl.

Exemplary substituents wherein at least two R¹ groups have been joined together include the following:

Particular disclosed embodiments concern cyclic compounds comprising a five-membered heteroatom-containing skeleton having a formula selected from those provided below.

With reference to Formulas 4-6, R¹ and n are as recited herein, and each X independently may be selected from carbon, oxygen, nitrogen, and sulfur.

In yet other embodiments, the cyclic compound comprising a five-membered heteroatom-containing skeleton may have any one of the following formulas

wherein R¹ is as recited herein.

Exemplary compounds are provided below.

TABLE 1 Exemplary Compounds

Particular embodiments concern cyclic compounds comprising a six-membered heteroatom-containing skeleton having any one of the formulas provided below:

wherein R² and n are as recited herein, Z may be selected from carbon and nitrogen, Y may be selected from nitrogen and oxygen, and each X independently may be selected from nitrogen and carbon. A person of ordinary skill in the art will recognize that the dashed lines indicate variable bonds which may or may not be present, depending on the valency of the atom to which each variable bond is attached. For example, if the variable bond indicated in Formula 11 is present, X typically is carbon, as a carbon atom can accommodate four bonds. X may be nitrogen in such a compound; however, a person of ordinary skill in the art would recognize that the nitrogen atom would be positively charged due to the fact that its lone pairs are used to accommodate a fourth bond.

Exemplary compounds are provided below solely as illustrative examples.

TABLE 2 Exemplary Compounds

Particular embodiments concern compounds comprising an all-carbon, steroidal skeleton having a formula as illustrated below.

An exemplary compound is provided below.

In other disclosed embodiments of the compound comprising an all-carbon skeleton, ring A is not connected with ring B and exists as an aryl compound having a formula illustrated below.

Exemplary compounds are illustrated below.

Other compounds suitable for use in the methods disclosed herein are provided below in Tables 3, 4, 5, and 6. Any of the compounds provided in Tables 3-6, and any other compounds disclosed herein, can be made using synthetic methods well known in the art of chemical synthesis.

TABLE 3

R¹ R¹′ R² R³ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ H NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)OH NHR³, or N[(CH₂)₀₋₅CH₃]₂ Halo H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ H NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)OH NHR³, or N[(CH₂)₀₋₅CH₃]₂ OH H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —O(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NH₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ NO₂ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ CF₃ H or —(CH₂)₀₋₅CH₃ C(O)NH₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NHPh NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NH-4-morpholinylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NH-4-piperazinyllphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃ C(O)NH-3-thiomethylphenyl NHR³, or N[(CH₂)₀₋₅CH₃]₂ H H or —(CH₂)₀₋₅CH₃

TABLE 4

R¹ R² C(O)NH₂ —Ph—Halo C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—Halo C(O)NHPh —Ph—Halo C(O)NH-2,4-fluorophenyl —Ph—Halo C(O)NH-4-fluorophenyl —Ph—Halo C(O)NH-3-fluorophenyl —Ph—Halo C(O)NH-4- —Ph—Halo morpholinylphenyl C(O)NH-4- —Ph—Halo piperazinyllphenyl C(O)NH-3- —Ph—Halo thiomethylphenyl H —Ph—Halo C(O)O(CH₂)₀₋₅CH₃ —Ph—Halo C(O)OH —Ph—Halo Ph—OH —Ph—Halo Ph—NH₂ —Ph—Halo Ph—Halo —Ph—Halo Ph—O(CH₂)₀₋₅CH₃ —Ph—Halo Ph—N[(CH2)₀₋₅CH3]2 —Ph—Halo C(O)NH-2 —Ph—OH C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—OH C(O)NHPh —Ph—OH C(O)NH-2,4-fluorophenyl —Ph—OH C(O)NH-4-fluorophenyl —Ph—OH C(O)NH-3-fluorophenyl —Ph—OH C(O)NH-4- —Ph—OH morpholinylphenyl C(O)NH-4- —Ph—OH piperazinyllphenyl C(O)NH-3- —Ph—OH thiomethylphenyl H —Ph—OH C(O)O(CH₂)₀₋₅CH₃ —Ph—OH C(O)OH —Ph—OH Ph—OH —Ph—OH Ph—NH₂ —Ph—OH Ph—Halo —Ph—OH Ph—O(CH₂)₀₋₅CH₃ —Ph—OH Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—OH C(O)NH₂ —Ph—O(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—O(CH₂)₀₋₅CH₃ C(O)NHPh —Ph—O(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl —Ph—O(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl —Ph—O(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl —Ph—O(CH₂)₀₋₅CH₃ C(O)NH-4- —Ph—O(CH₂)₀₋₅CH₃ morpholinylphenyl C(O)NH-4- —Ph—O(CH₂)₀₋₅CH₃ piperazinyllphenyl C(O)NH-3- —Ph—O(CH₂)₀₋₅CH₃ thiomethylphenyl H —Ph—O(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ —Ph—O(CH₂)₀₋₅CH₃ C(O)OH —Ph—O(CH₂)₀₋₅CH₃ Ph—OH —Ph—O(CH₂)₀₋₅CH₃ Ph—NH₂ —Ph—O(CH₂)₀₋₅CH₃ Ph—Halo —Ph—O(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ —Ph—O(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—O(CH₂)₀₋₅CH₃ C(O)NH₂ —Ph—NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—NH₂ C(O)NHPh —Ph—NH₂ C(O)NH-2,4-fluorophenyl —Ph—NH₂ C(O)NH-4-fluorophenyl —Ph—NH₂ C(O)NH-3-fluorophenyl —Ph—NH₂ C(O)NH-4- —Ph—NH₂ morpholinylphenyl C(O)NH-4- —Ph—NH₂ piperazinyllphenyl C(O)NH-3- —Ph—NH₂ thiomethylphenyl H —Ph—NH₂ C(O)O(CH₂)₀₋₅CH₃ —Ph—NH₂ C(O)OH —Ph—NH₂ Ph—OH —Ph—NH₂ Ph—NH₂ —Ph—NH₂ Ph—Halo —Ph—NH₂ Ph—O(CH₂)₀₋₅CH₃ —Ph—NH₂ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—NH₂ C(O)NH₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NHPh —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-2,4-fluorophenyl —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-fluorophenyl —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-fluorophenyl —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH-4- —Ph—N[(CH₂)₀₋₅CH₃]₂ morpholinylphenyl C(O)NH-4- —Ph—N[(CH₂)₀₋₅CH₃]₂ piperazinyllphenyl C(O)NH-3- —Ph—N[(CH₂)₀₋₅CH₃]₂ thiomethylphenyl H —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)OH —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—OH —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—NH₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—Halo —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—O(CH₂)₀₋₅CH₃ —Ph—N[(CH₂)₀₋₅CH₃]₂ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ —Ph—(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—(CH₂)₀₋₅CH₃ C(O)NHPh —Ph—(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl —Ph—(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl —Ph—(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl —Ph—(CH₂)₀₋₅CH₃ C(O)NH-4- —Ph—(CH₂)₀₋₅CH₃ morpholinylphenyl C(O)NH-4- —Ph—(CH₂)₀₋₅CH₃ piperazinyllphenyl C(O)NH-3- —Ph—(CH₂)₀₋₅CH₃ thiomethylphenyl H —Ph—(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ —Ph—(CH₂)₀₋₅CH₃ C(O)OH —Ph—(CH₂)₀₋₅CH₃ Ph—OH —Ph—(CH₂)₀₋₅CH₃ Ph—NH₂ —Ph—(CH₂)₀₋₅CH₃ Ph—Halo —Ph—(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ —Ph—(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—(CH₂)₀₋₅CH₃ C(O)NH₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NHPh —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-2,4-fluorophenyl —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-fluorophenyl —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-fluorophenyl —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH-4- —Ph—S[(CH₂)₀₋₅CH₃]₂ morpholinylphenyl C(O)NH-4- —Ph—S[(CH₂)₀₋₅CH₃]₂ piperazinyllphenyl C(O)NH-3- —Ph—S[(CH₂)₀₋₅CH₃]₂ thiomethylphenyl H —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)OH —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—OH —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—NH₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—Halo —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—O(CH₂)₀₋₅CH₃ —Ph—S[(CH₂)₀₋₅CH₃]₂ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ —Ph—NO₂ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—NO₂ C(O)NHPh —Ph—NO₂ C(O)NH-2,4-fluorophenyl —Ph—NO₂ C(O)NH-4-fluorophenyl —Ph—NO₂ C(O)NH-3-fluorophenyl —Ph—NO₂ C(O)NH-4- —Ph—NO₂ morpholinylphenyl C(O)NH-4- —Ph—NO₂ piperazinyllphenyl C(O)NH-3- —Ph—NO₂ thiomethylphenyl H —Ph—NO₂ C(O)O(CH₂)₀₋₅CH₃ —Ph—NO₂ C(O)OH —Ph—NO₂ Ph—OH —Ph—NO₂ Ph—NH₂ —Ph—NO₂ Ph—Halo —Ph—NO₂ Ph—O(CH₂)₀₋₅CH₃ —Ph—NO₂ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—NO₂ C(O)NH₂ —Ph—CF₃ C(O)N[(CH₂)₀₋₅CH₃]₂ —Ph—CF₃ C(O)NHPh —Ph—CF₃ C(O)NH-2,4-fluorophenyl —Ph—CF₃ C(O)NH-4-fluorophenyl —Ph—CF₃ C(O)NH-3-fluorophenyl —Ph—CF₃ C(O)NH-4- —Ph—CF₃ morpholinylphenyl C(O)NH-4- —Ph—CF₃ piperazinyllphenyl C(O)NH-3- —Ph—CF₃ thiomethylphenyl H —Ph—CF₃ C(O)O(CH₂)₀₋₅CH₃ —Ph—CF₃ C(O)OH —Ph—CF₃ Ph—OH —Ph—CF₃ Ph—NH₂ —Ph—CF₃ Ph—Halo —Ph—CF₃ Ph—O(CH₂)₀₋₅CH₃ —Ph—CF₃ Ph—N[(CH₂)₀₋₅CH₃]₂ —Ph—CF₃ C(O)NH₂ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)N[(CH₂)₀₋₅CH₃]₂ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NHPh NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NH-2,4-fluorophenyl NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NH-4-fluorophenyl NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NH-3-fluorophenyl NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NH-4- NH₂, NH(CH₂)₀₋₅CH₃, or morpholinylphenyl N[(CH₂)₀₋₅CH₃]₂ C(O)NH-4- NH₂, NH(CH₂)₀₋₅CH₃, or piperazinyllphenyl N[(CH₂)₀₋₅CH₃]₂ C(O)NH-3- NH₂, NH(CH₂)₀₋₅CH₃, or thiomethylphenyl N[(CH₂)₀₋₅CH₃]₂ H NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)OH NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—OH NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—NH2 NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—Halo NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—O(CH₂)₀₋₅CH₃ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ Ph—N[(CH₂)₀₋₅CH₃]₂ NH₂, NH(CH₂)₀₋₅CH₃, or N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NHPh NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-4- NHC(O)H or morpholinylphenyl NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-4- NHC(O)H or piperazinyllphenyl NHC(O)(CH₂)₀₋₅CH₃ C(O)NH-3- NHC(O)H or thiomethylphenyl NHC(O)(CH₂)₀₋₅CH₃ H NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)OH NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph—OH NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph—NH₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph—Halo NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ C(O)NH₂ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ NHS(O)₂H or NHS(O)₂(CH2)₀₋₅CH₃ C(O)NHPh NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-4- NHS(O)₂H or morpholinylphenyl NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-4- NHS(O)₂H or piperazinyllphenyl NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH-3- NHS(O)₂H or thiomethylphenyl NHS(O)₂(CH₂)₀₋₅CH₃ H NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)OH NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—OH NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—NH₂ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—Halo NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ NHS(O)₂H or NHS(O)₂(CH₂)₀₋₅CH₃ C(O)NH₂ OH or —O(CH₂)₀₋₅CH₃ C(O)N[(CH2)₀₋₅CH3]2 OH or —O(CH₂)₀₋₅CH₃ C(O)NHPh OH or —O(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl OH or —O(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl OH or —O(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl OH or —O(CH₂)₀₋₅CH₃ C(O)NH-4- OH or —O(CH₂)₀₋₅CH₃ morpholinylphenyl C(O)NH-4- OH or —O(CH₂)₀₋₅CH₃ piperazinyllphenyl C(O)NH-3- OH or —O(CH₂)₀₋₅CH₃ thiomethylphenyl H OH or —O(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ OH or —O(CH₂)₀₋₅CH₃ C(O)OH OH or —O(CH₂)₀₋₅CH₃ Ph—OH OH or —O(CH₂)₀₋₅CH₃ Ph—NH₂ OH or —O(CH₂)₀₋₅CH₃ Ph—Halo OH or —O(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ OH or —O(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ OH or —O(CH₂)₀₋₅CH₃ C(O)NH₂ 1-pyrrolidyl C(O)N[(CH₂)₀₋₅CH₃]₂ 1-pyrrolidyl C(O)NHPh 1-pyrrolidyl C(O)NH-2,4-fluorophenyl 1-pyrrolidyl C(O)NH-4-fluorophenyl 1-pyrrolidyl C(O)NH-3-fluorophenyl 1-pyrrolidyl C(O)NH-4- 1-pyrrolidyl morpholinylphenyl C(O)NH-4- 1-pyrrolidyl piperazinyllphenyl C(O)NH-3- 1-pyrrolidyl thiomethylphenyl H 1-pyrrolidyl C(O)O(CH₂)₀₋₅CH₃ 1-pyrrolidyl C(O)OH 1-pyrrolidyl Ph—OH 1-pyrrolidyl Ph—NH₂ 1-pyrrolidyl Ph—Halo 1-pyrrolidyl Ph—O(CH₂)₀₋₅CH₃ 1-pyrrolidyl Ph—N[(CH₂)₀₋₅CH₃]₂ 1-pyrrolidyl C(O)NH₂ 1-piperidinyl C(O)N[(CH₂)₀₋₅CH₃]₂ 1-piperidinyl C(O)NHPh 1-piperidinyl C(O)NH-2,4-fluorophenyl 1-piperidinyl C(O)NH-4-fluorophenyl 1-piperidinyl C(O)NH-3-fluorophenyl 1-piperidinyl C(O)NH-4- 1-piperidinyl morpholinylphenyl C(O)NH-4- 1-piperidinyl piperazinyllphenyl C(O)NH-3- 1-piperidinyl thiomethylphenyl H 1-piperidinyl C(O)O(CH₂)₀₋₅CH₃ 1-piperidinyl C(O)OH 1-piperidinyl Ph—OH 1-piperidinyl Ph—NH₂ 1-piperidinyl Ph—Halo 1-piperidinyl Ph—O(CH₂)₀₋₅CH₃ 1-piperidinyl Ph—N[(CH₂)₀₋₅CH₃]₂ 1-piperidinyl C(O)NH₂ 4-morpholinyl C(O)N[(CH₂)₀₋₅CH₃]₂ 4-morpholinyl C(O)NHPh 4-morpholinyl C(O)NH-2,4-fluorophenyl 4-morpholinyl C(O)NH-4-fluorophenyl 4-morpholinyl C(O)NH-3-fluorophenyl 4-morpholinyl C(O)NH-4- 4-morpholinyl morpholinylphenyl C(O)NH-4- 4-morpholinyl piperazinyllphenyl C(O)NH-3- 4-morpholinyl thiomethylphenyl H 4-morpholinyl C(O)O(CH₂)₀₋₅CH₃ 4-morpholinyl C(O)OH 4-morpholinyl Ph—OH 4-morpholinyl Ph—NH₂ 4-morpholinyl Ph—Halo 4-morpholinyl Ph—O(CH₂)₀₋₅CH₃ 4-morpholinyl Ph—N[(CH₂)₀₋₅CH₃]₂ 4-morpholinyl C(O)NH₂ —(CH₂)₀₋₅CH₃ C(O)N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ C(O)NHPh —(CH₂)₀₋₅CH₃ C(O)NH-2,4-fluorophenyl —(CH₂)₀₋₅CH₃ C(O)NH-4-fluorophenyl —(CH₂)₀₋₅CH₃ C(O)NH-3-fluorophenyl —(CH₂)₀₋₅CH₃ C(O)NH-4- —(CH₂)₀₋₅CH₃ morpholinylphenyl C(O)NH-4- —(CH₂)₀₋₅CH₃ piperazinyllphenyl C(O)NH-3- —(CH₂)₀₋₅CH₃ thiomethylphenyl H —(CH₂)₀₋₅CH₃ C(O)O(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ C(O)OH —(CH₂)₀₋₅CH₃ Ph—OH —(CH₂)₀₋₅CH₃ Ph—NH₂ —(CH₂)₀₋₅CH₃ Ph—Halo —(CH₂)₀₋₅CH₃ Ph—O(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ Ph—N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ C(O)NH₂ H C(O)N[(CH₂)₀₋₅CH₃]₂ H C(O)NHPh H C(O)NH-2,4-fluorophenyl H C(O)NH-4-fluorophenyl H C(O)NH-3-fluorophenyl H C(O)NH-4- H morpholinylphenyl C(O)NH-4- H piperazinyllphenyl C(O)NH-3- H thiomethylphenyl H H C(O)O(CH₂)₀₋₅CH₃ H C(O)OH H Ph—OH H Ph—NH₂ H Ph—Halo H Ph—O(CH₂)₀₋₅CH₃ H Ph—N[(CH₂)₀₋₅CH₃]₂ H

TABLE 5

 

 

 

 

R¹ R² R³ R¹ R² R³ pyrrolidinyl H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or pyrrolidinyl H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ piperidinyl H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or piperidinyl H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ indole H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or indole H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ NH₂ H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or NH₂ H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ —NHPh H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or —NHPh H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ Ph H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or Ph H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ OPh H, NO₂, CN, or NH₂, NH(CH₂)₀₋₅CH₃, or OPh H, NO₂, CN, or OH SO₂CF₃ N[(CH₂)₀₋₅CH₃]₂ SO₂CF₃ pyrrolidinyl H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ pyrrolidinyl H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ piperidinyl H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ piperidinyl H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ indole H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ indole H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ NH₂ H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ NH₂ H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ —NHPh H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ —NHPh H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ Ph H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ Ph H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ OPh H, NO₂, CN, or NHC(O)H or NHC(O)(CH₂)₀₋₅CH₃ OPh H, NO₂, CN, or 1-pyrrolidyl SO₂CF₃ SO₂CF₃ pyrrolidinyl H, NO₂, CN, or NHS(O)₂H or pyrrolidinyl H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl piperidinyl H, NO₂, CN, or NHS(O)₂H or piperidinyl H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl indole H, NO₂, CN, or NHS(O)₂H or indole H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl NH₂ H, NO₂, CN, or NHS(O)₂H or NH₂ H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl —NHPh H, NO₂, CN, or NHS(O)₂H or —NHPh H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl Ph H, NO₂, CN, or NHS(O)₂H or Ph H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl OPh H, NO₂, CN, or NHS(O)₂H or OPh H, NO₂, CN, or 1- SO₂CF₃ NHS(O)₂(CH₂)₀₋₅CH₃ SO₂CF₃ piperidinyl pyrrolidinyl H, NO₂, CN, or H pyrrolidinyl H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ piperidinyl H, NO₂, CN, or H piperidinyl H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ indole H, NO₂, CN, or H indole H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ NH₂ H, NO₂, CN, or H NH₂ H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ —NHPh H, NO₂, CN, or H —NHPh H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ Ph H, NO₂, CN, or H Ph H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ OPh H, NO₂, CN, or H OPh H, NO₂, CN, or Ph SO₂CF₃ SO₂CF₃ pyrrolidinyl H, NO₂, CN, or —NHPh pyrrolidinyl H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃ piperidinyl H, NO₂, CN, or —NHPh piperidinyl H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃ indole H, NO₂, CN, or —NHPh indole H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃ NH₂ H, NO₂, CN, or —NHPh NH₂ H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃ —NHPh H, NO₂, CN, or —NHPh —NHPh H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃ Ph H, NO₂, CN, or —NHPh Ph H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃ OPh H, NO₂, CN, or —NHPh OPh H, NO₂, CN, or —Ph-Halo SO₂CF₃ SO₂CF₃

TABLE 6

R¹ R³ R² (Selected from on of the following) OC(O)NH₂ Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ Halo C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ Halo C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ OH C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ OH C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ O(CH₂)₀₋₅CH₃ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ O(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ NH₂ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ NH₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ N[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ —(CH₂)₀₋₅CH₃ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ —(CH₂)₀₋₅CH₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ S[(CH₂)₀₋₅CH₃]₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ NO₂ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ NO₂ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)NH₂ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)N[(CH₂)₀₋₅CH₃]₂ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OH CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OP(O)(OH)₂ CF₃ C(O)NH₂₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)C(CH₃)₃ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)OC(O)NMe₂ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)O(CH₂)₀₋₅CH₃ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ OC(O)(CH₂)₀₋₅CH₃ CF₃ C(O)NH₂ C(O)N[(CH₂)₀₋₅CH₃]₂ C(O)O(CH₂)₀₋₅CH₃ Certain embodiments can use any one or more of the following compounds:

III. Methods of Use

i. Methods of Treating Muscular Dystrophy

The α7β1 integrin has been shown to be a major modifier of disease progression in patients with muscular dystrophy. Increased expression of the α7 integrin in muscle can alleviate muscle disease in mouse models of muscular dystrophy. By use of a muscle cell-based assay, the inventors identified molecules that up-regulate α7β1 integrin expression in muscle: laminin-111; valproic acid; ciclopirox ethanolamine; deferoxamine; 2,2-dipyridyl; 5α-cholestan-3β-ol-6-one; Compound ID#1001; Compound ID#1002; Compound ID #1003; and analogs of cholestan. Based on these observations, methods of treatment of muscular dystrophy by increasing the expression or activity of α7β1 integrin with additional suitable compounds are disclosed.

In particular, methods are disclosed herein for treating muscular dystrophy, such as DMD, FCMD, LGMD, FHMD, BMD, MDC1A or MDC1D. In one example, the method includes administering an effective amount of a α7β1 integrin modulatory agent to a subject with muscular dystrophy or suspected of having or developing muscular dystrophy, in which the agent increases the biological activity or expression of α7β1 integrin and thereby, treating the muscular dystrophy in the subject. In some example, the method of treatment inhibits or reduces one or more signs or symptoms associated with muscular dystrophy in the subject.

In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In some examples, an analog is synthesized according to the synthesis pathway shown in the Schemes below. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. For examples, in some examples, the α7β1 integrin modulatory agent includes one or more molecules provided by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.

The disclosed α7β1 integrin modulatory agents can alter the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).

In a particular example, the subject is a human.

In additional aspects, the method involves selecting a subject with muscular dystrophy. In some example, a subject is selected for treatment following diagnosing the subject with muscular dystrophy. For example, the method can include diagnosing the subject as suffering from muscular dystrophy, such as DMD, MDC1A, MDC1D, LGMD, DMD, FCMD or FHMD.

Methods of diagnosing a subject with muscular dystrophy are known to those of skill in the art and include, but are not limited to, muscle biopsies and measuring serum creatine kinase levels. Additionally, alterations in biomarker known to be associated with muscular dystrophy may be detected by measuring such levels in serum or urine sample.

In a further implementation, the method involves diagnosing the subject as suffering from a disease, disorder, or condition characterized by a mutation in the gene encoding α7 integrin. In another implementation, the method involves diagnosing the subject as suffering from a disease, disorder, or condition characterized by a decreased level of α7 integrin expression.

Alterations in the expression can be measured at the nucleic acid level (such as by real time quantitative polymerase chain reaction or microarray analysis) or at the protein level (such as by Western blot analysis or ELISA). These methods are known to those of skill in the art.

In some examples, following the measurement of the expression levels of α7 integrin expression or serum creatine kinase levels, the assay results, findings, diagnoses, predictions and/or treatment recommendations are recorded and communicated to technicians, physicians and/or patients, for example. In certain embodiments, computers are used to communicate such information to interested parties, such as, patients and/or the attending physicians. The therapy selected for administered is then based upon these results.

In one embodiment, the results and/or related information is communicated to the subject by the subject's treating physician. Alternatively, the results may be communicated directly to a test subject by any means of communication, including writing, such as by providing a written report, electronic forms of communication, such as email, or telephone. Communication may be facilitated by use of a computer, such as in case of email communications. In certain embodiments, the communication containing results of a diagnostic test and/or conclusions drawn from and/or treatment recommendations based on the test, may be generated and delivered automatically to the subject using a combination of computer hardware and software which will be familiar to artisans skilled in telecommunications. One example of a healthcare-oriented communications system is described in U.S. Pat. No. 6,283,761; however, the present disclosure is not limited to methods which utilize this particular communications system. In certain embodiments of the methods of the disclosure, all or some of the method steps, including the assaying of samples, diagnosing of diseases, and communicating of assay results or diagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

In several embodiments, identification of a subject as having muscular dystrophy, such as DMD, LGMD, FHMD, BMD, FCMD, MDC1D or MDC1A, results in the physician treating the subject, such as prescribing one or more disclosed α7β1 agents for inhibiting or delaying one or more signs and symptoms associated with muscular dystrophy. In additional embodiments, the dose or dosing regimen is modified based on the information obtained using the methods disclosed herein.

ii. Methods of Enhancing Muscle Regeneration, Repair, or Maintenance

Also disclosed are methods of enhancing muscle regeneration, repair or maintenance in a subject. In some examples, the method includes administering an effective amount of an α7β1 integrin modulatory agent to a subject in need of muscle regeneration, repair or maintenance, wherein the α7β1 integrin modulatory agent includes a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.

In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In some examples, an analog is synthesized according to the synthesis pathway provided in the Examples below. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. For examples, in some examples, the α7β1 integrin modulatory agent includes one or more molecules provided in a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.

The disclosed α7β1 integrin modulatory agents can increase the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).

Muscle regeneration may benefit, for example, geriatric or other patient populations with reduced muscle repair capability, or simply speed the muscle repair process for otherwise physiologically unimpaired patients. In particular implementations, administration of a α7β1 integrin modulatory agent can aid muscle repair, or reduction of muscle damage, in athletes or others having activity-induced muscle injury or damage. In yet further implementations, muscle repair in patients suffering from muscle damage, such as through accident or injury, can be augmented by administration of a α7β1 integrin modulatory agent.

In some examples, α7β1 modulatory agent is administered prior to the subject experiencing muscle damage or disease. In some examples, the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.

In some examples, the method further includes selecting a subject in need of enhancing muscle regeneration, repair, or maintenance. For example, in some instances, selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject. Methods for diagnosing and selecting a subject in need of muscle regeneration, repair or maintenance are known to those of ordinary skill in the art and include those provided described herein (including those in the Methods of Treatment of Muscular Dystrophy). As stated above, subjects may be selected based upon their life style (e.g., engaged in moderate to intense exercise or physical activities), age (e.g., elderly population at more risk of experiencing muscle degeneration or injury) or pre-disposition to muscle degeneration or injury (e.g., genetics or previous muscle injury).

iii. Methods of Prospectively Preventing or Reducing Muscle Injury or Damage

Also disclosed are methods prospectively preventing or reducing muscle injury or damage in a subject. In some embodiments, the method includes administering an effective amount of an α7β1 integrin modulatory agent to the subject wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof, wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby prospectively preventing or reducing muscle injury or damage in the subject.

In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. For examples, in some examples, the α7β1 integrin modulatory agent includes one or more molecules provided by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.

An exemplary synthetic scheme for making certain α7β1 integrin modulatory agent disclosed herein is provided in Schemes 1 and 2 as illustrated in FIG. 12. A person of ordinary skill in the art would recognize that derivatives of such compounds can be obtained using methods known in the art, such as functionalizing the core structure using suitable reagents and conditions.

Exemplary groups that can be modified to produce various analogs are indicated in the Schemes shown in FIG. 12; and in some embodiments, the core of the molecule can be modified to include one or more additional heteroatoms and/or to replace an existing heteroatom with a different suitable heteroatom.

The disclosed α7β1 integrin modulatory agents can increase the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).

In some examples, the method further includes selecting a subject at risk for developing a muscle injury or damage. In some examples, the α7β1 integrin modulatory agent is administered to a subject prior to the subject exercising.

In some examples, the method further includes selecting a subject at risk for developing a muscle injury or damage. Methods for selecting such s subject are known to those of ordinary skill in the art and include those provided described herein. As stated above, subjects may be selected based upon their life style (e.g., engaged in moderate to intense exercise or physical activities), age (elderly population at more risk of experiencing muscle degeneration or injury) or pre-disposition to muscle degeneration or injury (e.g., genetics or previous muscle injury).

iv. Methods of Enhancing α7β1 Integrin Expression

Also disclosed herein are methods of enhancing α7β1 integrin expression. In some examples, these methods include contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent comprises a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18, or a combination thereof and increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression. In some examples, the cell is a muscle cell, such as a skeletal muscle cell. In some examples, the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.

In some examples, the α7β1 integrin modulatory agent includes one or more of the following molecules: a compound encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18. In further examples, the α7β1 integrin modulatory agent is an analog/derivative of any of the disclosed α7β1 integrin modulatory agents which may be designed and synthesized according to the chemical principles known to one of ordinary skill in the art and identified as a α7β1 integrin modulatory agent by methods known to those of ordinary skill in the art, including the muscle cell based assay described Example 1. In some examples, the α7β1 integrin modulatory agent includes one or more molecules encompassed by any one of Formulas 1-16, or provided by any one of Tables 1-16 and 18.

In some examples, the disclosed α7β1 integrin modulatory agents can increase the expression of nucleic acid sequences (such as DNA, cDNA, or mRNAs) and proteins of α7β1 integrin. An increase in the expression or activity does not need to be 100% for the agent to be effective. For example, an agent can increase the expression or biological activity by a desired amount, for example by at least 10%, for example at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, including about 15% to about 98%, about 30% to about 95%, about 40% to about 80%, about 50% to about 70%, including about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98% or about 100%, as compared to activity or expression in a control. Methods of assessing α7β1 integrin expression and activity are known to those of ordinary skill in the art, including those described in the Examples below (e.g., Western blot and ELISA assay with commercially available antibodies).

Administration of an Effective Amount of an α7β1 Integrin Modulatory Agent

For any of the disclosed methods, an effective amount of α7β1 integrin modulatory agent is one when administered by a particular route and concentration induces the desired response (e.g., treatment of muscular dystrophy, enhancing muscle regeneration, repair or maintenance, preventing or reducing muscle injury or damage, or enhancing α7β1 integrin expression).

i. Administration Routes, Formulations and Concentrations

Methods of administration of the disclosed α7β1 integrin modulatory agents are routine, and can be determined by a skilled clinician. The disclosed α7β1 integrin modulatory agents or other therapeutic substance are in general administered topically, nasally, intravenously, orally, intracranially, intramuscularly, parenterally or as implants, but even rectal or vaginal use is possible in principle. The disclosed α7β1 integrin modulatory agents also may be administered to a subject using a combination of these techniques.

Suitable solid or liquid pharmaceutical preparation forms are, for example, aerosols, (micro)capsules, creams, drops, drops or injectable solution in ampoule form, emulsions, granules, powders, suppositories, suspensions, syrups, tablets, coated tablets, and also preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as binders, coating agents, disintegrants, flavorings, lubricants, solubilizers, sweeteners, or swelling agents are customarily used as described above. The pharmaceutical agents are suitable for use in a variety of drug delivery systems. For a brief review of various methods for drug delivery, see Langer, “New Methods of Drug Delivery,” Science 249:1527-1533 (1990), incorporated by reference herein to the extent not inconsistent with the present disclosure.

The disclosed α7β1 integrin modulatory agents or other therapeutic agents of the present disclosure can be formulated into therapeutically-active pharmaceutical agents that can be administered to a subject parenterally or orally. Parenteral administration routes include, but are not limited to epidermal, intraarterial, intramuscular (IM and depot IM), intraperitoneal (IP), intravenous (IV), intrasternal injection or infusion techniques, intranasal (inhalation), intrathecal, injection into the stomach, subcutaneous injections (subcutaneous (SQ and depot SQ), transdermal, topical, and ophthalmic.

The disclosed α7β1 integrin modulatory agents or other therapeutic agents can be mixed or combined with a suitable pharmaceutically acceptable excipients to prepare pharmaceutical agents. Pharmaceutically acceptable excipients include, but are not limited to, alumina, aluminum stearate, buffers (such as phosphates), glycine, ion exchangers (such as to help control release of charged substances), lecithin, partial glyceride mixtures of saturated vegetable fatty acids, potassium sorbate, serum proteins (such as human serum albumin), sorbic acid, water, salts or electrolytes such as cellulose-based substances, colloidal silica, disodium hydrogen phosphate, magnesium trisilicate, polyacrylates, polyalkylene glycols, such as polyethylene glycol, polyethylene-polyoxypropylene-block polymers, polyvinyl pyrrolidone, potassium hydrogen phosphate, protamine sulfate, group 1 halide salts such as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat, and zinc salts, for example. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers.

Upon mixing or addition of one or more disclosed α7β1 integrin modulatory agents and/or or other therapeutic agents, the resulting mixture may be a solid, solution, suspension, emulsion, or the like. These may be prepared according to methods known to those of ordinary skill in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the agent in the selected carrier. Pharmaceutical carriers suitable for administration of the disclosed α7β1 integrin modulatory agents or other therapeutic agents include any such carriers known to be suitable for the particular mode of administration. In addition, the disclosed α7β1 integrin modulatory agents or other therapeutic substance can also be mixed with other inactive or active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action.

Methods for solubilizing may be used where the agents exhibit insufficient solubility in a carrier. Such methods are known and include, but are not limited to, dissolution in aqueous sodium bicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), and using surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Del.).

The disclosed α7β1 integrin modulatory agents or other therapeutic agents can be prepared with carriers that protect them against rapid elimination from the body, such as coatings or time-release formulations. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. A disclosed α7β1 integrin modulatory agents or other therapeutic agent is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect, typically in an amount to avoid undesired side effects, on the treated subject. The therapeutically effective concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated condition. For example, mouse models of muscular dystrophy may be used to determine effective amounts or concentrations that can then be translated to other subjects, such as humans, as known in the art.

Injectable solutions or suspensions can be formulated, using suitable non-toxic, parenterally-acceptable diluents or solvents, such as 1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer's solution, saline solution, or water; or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid; a naturally occurring vegetable oil such as coconut oil, cottonseed oil, peanut oil, sesame oil, and the like; glycerine; polyethylene glycol; propylene glycol; or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; buffers such as acetates, citrates, and phosphates; chelating agents such as ethylenediaminetetraacetic acid (EDTA); agents for the adjustment of tonicity such as sodium chloride and dextrose; and combinations thereof. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required. Where administered intravenously, suitable carriers include physiological saline, phosphate-buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions, including tissue-targeted liposomes, may also be suitable as pharmaceutically acceptable carriers.

For topical application, one or more disclosed α7β1 integrin modulatory agents, or other therapeutic agent may be made up into a cream, lotion, ointment, solution, or suspension in a suitable aqueous or non-aqueous carrier. Topical application can also be accomplished by transdermal patches or bandages which include the therapeutic substance. Additives can also be included, e.g., buffers such as sodium metabisulphite or disodium edetate; preservatives such as bactericidal and fungicidal agents, including phenyl mercuric acetate or nitrate, benzalkonium chloride, or chlorhexidine; and thickening agents, such as hypromellose.

If the disclosed α7β1 integrin modulatory agent, or other therapeutic agent is administered orally as a suspension, the pharmaceutical agents can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain a suspending agent, such as alginic acid or sodium alginate, bulking agent, such as microcrystalline cellulose, a viscosity enhancer, such as methylcellulose, and sweeteners/flavoring agents. Oral liquid preparations can contain conventional additives such as suspending agents, e.g., gelatin, glucose syrup, hydrogenated edible fats, methyl cellulose, sorbitol, and syrup; emulsifying agents, e.g., acacia, lecithin, or sorbitan monooleate; non-aqueous carriers (including edible oils), e.g., almond oil, fractionated coconut oil, oily esters such as glycerine, propylene glycol, or ethyl alcohol; preservatives such as methyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired, conventional flavoring or coloring agents. When formulated as immediate release tablets, these agents can contain dicalcium phosphate, lactose, magnesium stearate, microcrystalline cellulose, and starch and/or other binders, diluents, disintegrants, excipients, extenders, and lubricants.

If oral administration is desired, one or more disclosed α7β1 integrin modulatory agents, or other therapeutic substances can be provided in a composition that protects it from the acidic environment of the stomach. For example, he disclosed α7β1 integrin modulatory agents or other therapeutic agents can be formulated with an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The disclosed α7β1 integrin modulatory agents, or other therapeutic agent can also be formulated in combination with an antacid or other such ingredient.

Oral compositions generally include an inert diluent or an edible carrier and can be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, one or more of the disclosed α7β1 integrin modulatory agents, or other therapeutic substances can be incorporated with excipients and used in the form of capsules, tablets, or troches. Pharmaceutically compatible adjuvant materials or binding agents can be included as part of the composition.

The capsules, pills, tablets, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, acacia, corn starch, gelatin, gum tragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calcium phosphate, glycine, lactose, microcrystalline cellulose, or starch; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate, polyethylene glycol, silica, or talc; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; disintegrants such as potato starch; dispersing or wetting agents such as sodium lauryl sulfate; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier, such as a fatty oil. In addition, dosage unit forms can contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. One or more of the disclosed α7β1 integrin modulatory agents, or other therapeutic agent can also be administered as a component of an elixir, suspension, syrup, wafer, tea, chewing gum, or the like. A syrup may contain, in addition to the active compounds, sucrose or glycerin as a sweetening agent and certain preservatives, dyes and colorings, and flavors.

When administered orally, the compounds can be administered in usual dosage forms for oral administration. These dosage forms include the usual solid unit dosage forms of tablets and capsules as well as liquid dosage forms such as solutions, suspensions, and elixirs. When the solid dosage forms are used, they can be of the sustained release type so that the compounds need to be administered less frequently.

In some examples, one or more of the disclosed α7β1 integrin modulatory agents and/or a therapeutic agent is injected into the stomach of a subject is incorporated systemically in the subject, such as in diverse muscle groups. Examples of methods and compositions for administering therapeutic substances which include proteins include those discussed in Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems 2ed. (2005); Mahato, Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids (2004); McNally, Protein Formulation and Delivery, 2ed. (2007); and Kumar et al., “Novel Delivery Technologies for Protein and Peptide Therapeutics,” Current Pharm. Biotech., 7:261-276 (2006); each of which is incorporated by reference herein to the extent not inconsistent with the present disclosure.

In some implementations, the effective amount of one or more of the disclosed α7β1 integrin modulatory agents is administered as a single dose per time period, such as every three or four months, month, week, or day, or it can be divided into at least two unit dosages for administration over a period. Treatment may be continued as long as necessary to achieve the desired results. For instance, treatment may continue for about 3 or 4 weeks up to about 12-24 months or longer, including ongoing treatment. The compound can also be administered in several doses intermittently, such as every few days (for example, at least about every two, three, four, five, or ten days) or every few weeks (for example at least about every two, three, four, five, or ten weeks).

Particular dosage regimens can be tailored to a particular subject, condition to be treated, or desired result. For example, when the methods of the present disclosure are used to treat muscular dystrophy or similar conditions, an initial treatment regimen can be applied to arrest the condition. Such initial treatment regimen may include administering a higher dosage of one or more of the disclosed α7β1 integrin modulatory agents, or administering such material more frequently, such as daily. After a desired therapeutic result has been obtained, such as a desired level of muscle regeneration, a second treatment regimen may be applied, such as administering a lower dosage of one or more of the disclosed α7β1 integrin modulatory agents or administering such material less frequently, such as monthly, bi-monthly, quarterly, or semi-annually. In such cases, the second regimen may serve as a “booster” to restore or maintain a desired level of muscle regeneration. Similar treatment regimens may be used for other subjects with reduced or impaired muscle regeneration capabilities, such as geriatric subjects.

When particular methods of the present disclosure are used to prevent or mitigate muscle damage, such as damage caused by exertion or injury, the subject is typically treated a sufficient period of time before the exertion or injury in order to provide therapeutic effect. For example, the subject may be treated at least about 24 hours before the expected activity or potential injury, such as at least about 48 hours, about 72 hours, about 1 week, about 2 weeks, about three weeks, or about 4 weeks or more prior.

When embodiments of the method of the present disclosure are used to prevent or treat a muscle injury, one or more of the disclosed α7β1 integrin modulatory agents or other therapeutic substance can be applied directly to, or proximately to, the area to be treated. For example, the substance can be injected into or near the area. In further examples, the substance can be applied topically to the area to be treated. Treatment is typically initiated prior to the injury to several weeks following the injury. In more specific implementations, the treatment is initiated between about 12 and about 72 hours following injury, such as between about 24 and about 48 hours following injury. In some cases, a single administration of the substance is effective to provide the desired therapeutic effect. In further examples, additional administrations are provided in order to achieve the desired therapeutic effect.

Amounts effective for various therapeutic treatments of the present disclosure may, of course, depend on the severity of the disease and the weight and general state of the subject, as well as the absorption, inactivation, and excretion rates of the therapeutically-active compound or component, the dosage schedule, and amount administered, as well as other factors known to those of ordinary skill in the art. It also should be apparent to one of ordinary skill in the art that the exact dosage and frequency of administration will depend on the particular α7β1 integrin modulatory agent, or other therapeutic substance being administered, the particular condition being treated, the severity of the condition being treated, the age, weight, general physical condition of the particular subject, and other medication the subject may be taking. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in vivo administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of particular disorders. For example, mouse models of muscular dystrophy may be used to determine effective dosages that can then be translated to dosage amount for other subjects, such as humans, as known in the art. Various considerations in dosage determination are described, e.g., in Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press (1990); and Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa. (1990), each of which is herein incorporated by reference to the extent not inconsistent with the present disclosure.

In specific examples, the one or more disclosed α7β1 integrin modulatory agents is administered to a subject in an amount sufficient to provide a dose of the agent of between about 10 fmol/g and about 500 nmol/g, such as between about 2 nmol/g and about 20 nmol/g or between about 2 nmol/g and about 10 nmol/g. In additional examples, the α7β1 integrin modulatory agent is administered to a subject in an amount sufficient to provide a dose of between about 0.01 μg/kg and about 1000 mg/kg or between about 0.1 mg/kg and about 1000 mg/kg, in particular examples this amount is provided per day or per week. In another example, the disclosed α7β1 integrin modulatory agent is administered to a subject in an amount sufficient to provide a dose of agent of between about 0.2 mg/kg and about 2 mg/kg. In further examples, the α7β1 integrin modulatory agent is administered to a subject in an amount sufficient to provide a concentration of α7β1 integrin modulatory agent in the administrated material of between about 5 nM and about 500 nM, such as between about 50 nM and about 200 nm, or about 100 nM. In other examples, the α7β1 integrin modulatory agent is administered to a subject between about 500 μg/ml and about 1 μg/ml, such as about 300 μg/ml and about 3 μg/ml, about 200 μg/ml and about 20 μg/ml, including 500 μg/ml, 400 μg/ml, 300 μg/ml, 250 μg/ml, 200 μg/ml, 150 μg/ml, 100 μg/ml, 50 μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.125 μg/ml, 2.5 μg/ml and 1.25 μg/ml.

ii. Desired Response

One or more disclosed α7β1 integrin modulatory agents and/or additional therapeutic agents are administered by a specific route and/or concentration to generate a desired response. In some examples, a desired response refers to an amount effective for lessening, ameliorating, eliminating, preventing, or inhibiting at least one symptom of a disease, disorder, or condition treated and may be empirically determined. In various embodiments of the present disclosure, a desired response is muscle regeneration, reductions or prevention of muscle degeneration, promotion of muscle maintenance, reduction or prevention of muscle injury or damage, reduction or prevention in one more signs or symptoms associated with muscular dystrophy.

In particular, indicators of muscular health, such as muscle cell regeneration, maintenance, or repair, can be assessed through various means, including monitoring markers of muscle regeneration, such as transcription factors such as Pax7, Pax3, MyoD, MRF4, and myogenin. For example, increased expression of such markers can indicate that muscle regeneration is occurring or has recently occurred. Markers of muscle regeneration, such as expression of embryonic myosin heavy chain (eMyHC), can also be used to gauge the extent of muscle regeneration, maintenance, or repair. For example, the presence of eMyHC can indicate that muscle regeneration has recently occurred in a subject.

Muscle cell regeneration, maintenance, or repair can also be monitored by determining the girth, or mean cross sectional area, of muscle cells or density of muscle fibers. Additional indicators of muscle condition include muscle weight and muscle protein content. Mitotic index (such as by measuring BrdU incorporation) and myogenesis can also be used to evaluate the extent of muscle regeneration.

In particular examples, the improvement in muscle condition, such as regeneration, compared with a control is at least about 10%, such as at least about 30%, or at least about 50% or more, including an at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, including a 10% to 90% decrease, 20% to 80% increase, 30% to 70% increase or a 40% to 60% increase (e.g., a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or more increase).

iii. Additional Treatments or Therapeutic Agents

In particular examples, prior to, during, or following administration of an effective amount of an agent that reduces or inhibits one or more signs or symptoms associated with muscular dystrophy, the subject can receive one or more other therapies. In one example, the subject receives one or more treatments prior to administration of a disclosed α7β1 modulatory agent. Examples of such therapies include, but are not limited to, laminin-111 protein therapy, which works to stabilize the sarcolemma and reduce muscle degeneration. In some examples, a source of muscle cells can be added to aid in muscle regeneration and repair. In some aspects of the present disclosure, satellite cells are administered to a subject in combination with laminin therapy. U.S. Patent Publication 2006/0014287, incorporated by reference herein to the extent not inconsistent with the present disclosure, provides methods of enriching a collection of cells in myogenic cells and administering those cells to a subject. In further aspects, stem cells, such as adipose-derived stem cells, are administered to the subject. Suitable methods of preparing and administering adipose-derived stem cells are disclosed in U.S. Patent Publication 2007/0025972, incorporated by reference herein to the extent not inconsistent with the present disclosure. Additional cellular materials, such as fibroblasts, can also be administered, in some examples.

Additional therapeutic agents include agents which enhance the effect of the disclosed α7β1 modulatory agents, such as a component of the extracellular matrix, such as an integrin, dystrophin, dystroglycan, utrophin, or a growth factor. In some examples, the additional therapeutic agent reduces or enhances expression of a substance that enhances the formation or maintenance of the extracellular matrix. In some examples, the additional substance can include aggrecan, angiostatin, cadherins, collagens (including collagen I, collagen III, or collagen IV), decorin, elastin, enactin, endostatin, fibrin, fibronectin, osteopontin, tenascin, thrombospondin, vitronectin, and combinations thereof. Biglycans, glycosaminoglycans (such as heparin), glycoproteins (such as dystroglycan), proteoglycans (such as heparan sulfate), and combinations thereof can also be administered.

In some examples, growth stimulants such as cytokines, polypeptides, and growth factors such as brain-derived neurotrophic factor (BDNF), CNF (ciliary neurotrophic factor), EGF (epidermal growth factor), FGF (fibroblast growth factor), glial growth factor (GGF), glial maturation factor (GMF) glial-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), insulin, insulin-like growth factors, kerotinocyte growth factor (KGF), nerve growth factor (NGF), neurotropin-3 and -4, PDGF (platelet-derived growth factor), vascular endothelial growth factor (VEGF), and combinations thereof may be administered with one of the disclosed methods.

IV. Clinical Trials

To obtain regulatory approval for the use of one or more of the disclosed α7β1 modulatory agents to treat a muscular disorder, clinical trials are performed. As is known in the art, clinical trials progress through phases of testing, which are identified as Phases I, II, III, and IV.

Initially the disclosed α7β1 modulatory agent is evaluated in a Phase I trial. Typically Phase I trials are used to determine the best mode of administration (for example, by pill or by injection), the frequency of administration, and the toxicity for the compounds. Phase I studies frequently include laboratory tests, such as blood tests and biopsies, to evaluate the effects of the potential therapeutic in the body of the patient. For a Phase I trial, a small group of patients with a muscular disorder are treated with a specific dose of a disclosed α7β1 modulatory agent. During the trial, the dose is typically increased group by group in order to determine the maximum tolerated dose (MTD) and the dose-limiting toxicities (DLT) associated with the compound. This process determines an appropriate dose to use in a subsequent Phase II trial.

A Phase II trial can be conducted to further evaluate the effectiveness and safety of the disclosed α7β1 modulatory agent. In Phase II trials, a disclosed α7β1 modulatory agent is administered to groups of patients with a muscular disorder using the dosage found to be effective in Phase I trials.

Phase III trials focus on determining how a disclosed α7β1 modulatory agent compares to the standard, or most widely accepted, treatment. In Phase III trials, patients are randomly assigned to one of two or more “arms”. In a trial with two arms, for example, one arm will receive the standard treatment (control group) and the other arm will receive a disclosed α7β1 modulatory agent treatment (investigational group).

Phase IV trials are used to further evaluate the long-term safety and effectiveness of a disclosed α7β1 modulatory agent. Phase IV trials are less common than Phase I, II and III trials and take place after a disclosed α7β1 modulatory agent has been approved for standard use.

Eligibility of Patients for Clinical Trials

Participant eligibility criteria can range from general (for example, age, sex, type of disease) to specific (for example, type and number of prior treatments, disease characteristics, blood cell counts, organ function). In one embodiment, eligible patients have been diagnosed with a muscular disorder. Eligibility criteria may also vary with trial phase. Patients eligible for clinical trials can also be chosen based on objective measurement of a muscular disorder and failure to respond to other muscular disorder treatments. For example, in Phase I and II trials, the criteria often exclude patients who may be at risk from the investigational treatment because of abnormal organ function or other factors. In Phase II and III trials additional criteria are often included regarding disease type and stage, and number and type of prior treatments.

Phase I trials usually include 15 to 30 participants for whom other treatment options have not been effective. Phase II trials typically include up to 100 participants who have already received drug therapy, but for whom the treatment has not been effective.

Participation in Phase III trials is often restricted based on the previous treatment received. Phase III trials usually include hundreds to thousands of participants. This large number of participants is necessary in order to determine whether there are true differences between the effectiveness of a disclosed α7β1 modulatory agent and the standard treatment. Phase III can include patients ranging from those newly diagnosed with a muscular disorder to those with re-occurring signs and/or symptoms associated with a muscular disorder or a muscular disorder that did not respond to prior treatment.

One skilled in the art will appreciate that clinical trials should be designed to be as inclusive as possible without making the study population too diverse to determine whether the treatment might be as effective on a more narrowly defined population. The more diverse the population included in the trial, the more applicable the results could be to the general population, particularly in Phase III trials. Selection of appropriate participants in each phase of clinical trial is considered to be within the ordinary skills of a worker in the art.

Assessment of Patients Prior to Treatment

Prior to commencement of the study, several measures known in the art can be used to first classify the patients. Patients can first be assessed, for example by determining serum creatine kinase (CK) levels or other indicators of a muscle disorder, such as increased levels of muscle inflammation, apoptosis, muscle loss, myotube hypertrophy, and/or decreased myofibers stability and cell survival.

Administration of a Disclosed α7β1 Modulatory Agent in Clinical Trials

A disclosed α7β1 modulatory agent is typically administered to the trial participants orally. A range of doses of the agent can be tested. Provided with information from preclinical testing, a skilled practitioner can readily determine appropriate dosages of agent for use in clinical trials. In one embodiment, a dose range is from about 100 μg/kg and about 5000 mg/kg of the subject's weight, such as 1 mg/kg and about 2000 mg/kg of the subject's weight, about 100 mg/kg and about 1500 mg/kg of the subject's weight, about 100 μg/kg and about 2000 mg/kg of the subject's weight, about 200 mg/kg and about 1000 mg/kg of the subject's weight, about 200 mg/kg and about 750 mg/kg of the subject's weight, about 250 mg/kg and about 500 mg/kg of the subject's weight, about 100 m and about 500 mM. In some embodiments, subjects are given a disclosed α7β1 modulatory agent orally at 10 to 60 mg/kg of body weight per day. For example, 10-15 mg/kg of a disclosed α7β1 modulatory agent is administered for two weeks and if well tolerated the dose is increased by 5-10 mg/kg/week to achieve optimal clinical response. In some examples, the daily dose does not exceed 60 mg/kg of body weight and is given for a minimum of 6 months with liver function monitored every two weeks to monthly.

Pharmacokinetic Monitoring

To fulfill Phase I criteria, distribution of the disclosed α7β1 modulatory agent is monitored, for example, by chemical analysis of samples, such as blood, collected at regular intervals. For example, samples can be taken at regular intervals up until about 72 hours after the start of treatment.

If analysis is not conducted immediately, the samples can be placed on dry ice after collection and subsequently transported to a freezer to be stored at −70° C. until analysis can be conducted. Samples can be prepared for analysis using standard techniques known in the art and the amount of the disclosed α7β1 modulatory agent present can be determined, for example, by high-performance liquid chromatography (HPLC). Pharmacokinetic data can be generated and analyzed in collaboration with an expert clinical pharmacologist and used to determine, for example, clearance, half-life and maximum plasma concentration.

Monitoring of Patient Outcome

The endpoint of a clinical trial is a measurable outcome that indicates the effectiveness of a compound under evaluation. The endpoint is established prior to the commencement of the trial and will vary depending on the type and phase of the clinical trial. Examples of endpoints include, for example, decline in serum CK levels, inflammation, apoptosis, and muscle loss. For example, at least a 10% reduction in serum CK levels indicates the patient is responsive to the treatment.

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.

EXAMPLES Example 1

This example demonstrates the results of quantitative real-time PCR used to assess Itga7, Itgb1, and Lama2 transcript levels in C2C12 myoblasts and myotubes treated for 24 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), 10 μM MLS001165937-01 (IED-937), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD (FIG. 1). FIG. 2 is a digital image of Western Blots and quantitative analysis of α7 Integrin and GAPDH protein levels in C2C12 myotubes treated for 48 hours with DMSO control, 10 μM MLS000683232-01 (IED-232), Hydroxylpropyl-Beta-Cyclodextrin (HPBCD) control, or 12 μM SU9516 in HPBCD. Bands were quantified using Image J software and then graphed as α7 Integrin protein levels relative to GAPDH protein levels. * denotes a significant difference in relative protein levels with **p<0.01.

Example 2

The additional structures provided below provide additional compounds that may be used in the methods disclosed herein increasing α7 integrin expression in muscle (see Table 7). In some examples, the analogs were made in 5 mg quantities, salt form (e.g., hydrochloride salt), as a dry powder, at an at least 90% purity as measured by HPLC. In some embodiments, physical properties (kinetic, solubility, PAMPA permeability, clog P) and metabolic stability (plasma and microsomal stability, metabolite identification) of all synthesized molecules, as well as measure pharmacokinetics properties, compound levels and distribution of selected compounds are determined. Besides systematic substitution for exploring selected chemotypes, classical medicinal chemistry parameters (MW, number of hydrogen donors and acceptors, tPSA, clogP, flexibility of the molecule) as well as in house-measured physical and metabolic properties of analog molecules are used to guide the selection of potent, efficacious and non-toxic compounds having suitable pharmacokinetic properties for use in the disclosed methods.

TABLE 7 Exemplary Compounds

  Potency: 1.122 Efficacy: 198.146 Compound 1

  Potency: 3.5481 Efficacy: 228.085 Compound 2

  Potency: 2.2387 Efficacy: 193.433 Compound 3

  Potency: 2.8184 Efficacy: 129.298 Compound 4

  Potency: 1.122 Efficacy: 92.4838 Compound 5

  Potency: 1.122 Efficacy: 128.12 Compound 6

  Potency: 4.4668 Efficacy: 94.9208 Compound 7

  Potency: 2.8184 Efficacy: 111.685 Compound 8

  Potency: 2.8184 Efficacy: 122.703 Compound 9

  Potency: 3.1623 Efficacy: 102.022 Compound 10

  Potency: 3.9811 Efficacy: 101.893 Compound 11

  Potency: 2.8184 Efficacy: 92.9057 Compound 12

  Potency: 4.4668 Efficacy: 121.996 Compound 13

  Potency: 2.2387 Efficacy: 84.7736 Compound 14

  Potency: 3.1623 Efficacy: 91.3808 Compound 15

  Potency: 2.2387 Efficacy: 115.349 Compound 16

  Potency: 2.5119 Efficacy: 96.1948 Compound 17

  Potency: 10 Efficacy: 1751.56 Compound 18

  Potency: 1.4125 Efficacy: 81.6138 Compound 19

  Potency: 3.5481 Efficacy: 101.579 Compound 20

  Potency: 5.6234 Efficacy: 92.2825 Compound 21

  Potency: 4.4668 Efficacy: 83.4252 Compound 22

  Potency: 3.9811 Efficacy: 85.1623 Compound 23

  Potency: 3.1623 Efficacy: 76.9771 Compound 24

  Potency: 2.2387 Efficacy: 82.8995 Compound 25

  Potency: 1.9953 Efficacy: 95.7363 Compound 26

  Potency: 1.9953 Efficacy: 81.8506 Compound 27

  Potency: 2.8184 Efficacy: 92.0171 Compound 28

  Potency: 3.9811 Efficacy: 81.1658 Compound 29

  Potency: 3.9811 Efficacy: 92.7977 Compound 30

  Potency: 2.5119 Efficacy: 85.4354 Compound 31

  Potency: 2.8184 Efficacy: 88.796 Compound 32

  Potency: 1.9953 Efficacy: 83.3258 Compound 33

  Potency: 1.4125 Efficacy: 70.4537 Compound 34

  Potency: 2.5119 Efficacy: 72.7515 Compound 35

  Potency: 3.1623 Efficacy: 75.7047 Compound 36

  Potency: 2.5119 Efficacy: 77.0458 Compound 37

  Potency: 4.4668 Efficacy: 75.5605 Compound 38

  Potency: 3.5481 Efficacy: 78.5404 Compound 39

  Potency: 14.1254 Efficacy: 436.691 Compound 40

  Potency: 14.1254 Efficacy: 447.862 Compound 41

  Potency: 14.1254 Efficacy: 475.262 Compound 42

  Potency: 3.9811 Efficacy: 148.01 Compound 43

  Potency: 11.2202 Efficacy: 328.277 Compound 44

  Potency: 14.1254 Efficacy: 426.535 Compound 45

  Potency: 7.9433 Efficacy: 231.415 Compound 46

  Potency: 5.6234 Efficacy: 141.317 Compound 47

  Potency: 7.0795 Efficacy: 214.145 Compound 48

  Potency: 5.6234 Efficacy: 150.998 Compound 49

  Potency: 7.9433 Efficacy: 181.323 Compound 50

  Potency: 10 Efficacy: 273.425 Compound 51

  Potency: 4.4668 Efficacy: 165.293 Compound 52

  Potency: 7.0795 Efficacy: 163.55 Compound 53

  Potency: 12.5893 Efficacy: 222.708 Compound 54

  Potency: 12.5893 Efficacy: 271.246 Compound 55

  Potency: 3.9811 Efficacy: 88.764 Compound 56

  Potency: 4.4668 Efficacy: 115.732 Compound 57

  Potency: 3.5481 Efficacy: 137.991 Compound 58

  Potency: 10 Efficacy: 188.981 Compound 59

  Potency: 4.4668 Efficacy: 128.904 Compound 60

  Potency: 5.0119 Efficacy: 140.322 Compound 61

  Potency: 7.0795 Efficacy: 186.257 Compound 62

  Potency: 10 Efficacy: 200.43 Compound 63

  Potency: 5.0119 Efficacy: 134.919 Compound 64

  Potency: 19.9526 Efficacy: 491.985 Compound 65

  Potency: 12.5893 Efficacy: 215.25 Compound 66

  Potency: 10 Efficacy: 157.616 Compound 67

  Potency: 12.5893 Efficacy: 239.617 Compound 68

  Potency: 3.9811 Efficacy: 90.5081 Compound 69

  Potency: 4.4668 Efficacy: 115.234 Compound 70

  Potency: 7.9433 Efficacy: 124.512 Compound 71

  Potency: 8.9125 Efficacy: 138.663 Compound 72

  Potency: 12.5893 Efficacy: 170.328 Compound 73

  Potency: 4.4668 Efficacy: 109.274 Compound 74

  Potency: 3.9811 Efficacy: 71.4431 Compound 75

  Potency: 6.3096 Efficacy: 114.425 Compound 76

  Potency: 4.4668 Efficacy: 121.35 Compound 77

  Potency: 10 Efficacy: 152.941 Compound 78

  Potency: 4.4668 Efficacy: 85.208 Compound 79

  Potency: 10 Efficacy: 175.516 Compound 80

  Potency: 12.5893 Efficacy: 213.226 Compound 81

  Potency: 3.1623 Efficacy: 96.4736 Compound 82

  Potency: 11.2202 Efficacy: 175.054 Compound 83

  Potency: 6.3096 Efficacy: 122.242 Compound 84

  Potency: 5.0119 Efficacy: 99.705 Compound 85

  Potency: 4.4668 Efficacy: 94.2018 Compound 86

  Potency: 5.0119 Efficacy: 66.4058 Compound 87

  Potency: 8.9125 Efficacy: 88.9864 Compound 88

  Potency: 5.0119 Efficacy: 86.1979 Compound 89

  Potency: 11.2202 Efficacy: 140.713 Compound 90

  Potency: 11.2202 Efficacy: 139.097 Compound 91

  Potency: 4.4668 Efficacy: 75.211 Compound 92

  Potency: 11.2202 Efficacy: 117.921 Compound 93

  Potency: 10 Efficacy: 128.349 Compound 94

  Potency: 7.0795 Efficacy: 98.2729 Compound 95

  Potency: 4.4668 Efficacy: 83.068 Compound 96

  Potency: 5.6234 Efficacy: 78.6154 Compound 97

  Potency: 10 Efficacy: 125.681 Compound 98

  Potency: 10 Efficacy: 121.073 Compound 99

  Potency: 3.9811 Efficacy: 95.5318 Compound 100

  Potency: 8.9125 Efficacy: 131.53 Compound 101

  Potency: 12.5893 Efficacy: 149.767 Compound 102

  Potency: 6.3096 Efficacy: 119.897 Compound 103

  Potency: 10 Efficacy: 137.595 Compound 104

  Potency: 10 Efficacy: 96.3714 Compound 105

  Potency: 7.0795 Efficacy: 81.3792 Compound 106

  Potency: 4.4668 Efficacy: 95.5906 Compound 107

  Potency: 12.5893 Efficacy: 139.967 Compound 108

  Potency: 12.5893 Efficacy: 166.495 Compound 109

  Potency: 12.5893 Efficacy: 180.783 Compound 110

  Potency: 7.0795 Efficacy: 99.2205 Compound 111

  Potency: 5.6234 Efficacy: 73.9187 Compound 112

  Potency: 10 Efficacy: 111.758 Compound 113

  Potency: 11.2202 Efficacy: 138.65 Compound 114

  Potency: 7.0795 Efficacy: 85.5436 Compound 115

  Potency: 8.9125 Efficacy: 104.576 Compound 116

  Potency: 7.9433 Efficacy: 88.998 Compound 117

  Potency: 5.0119 Efficacy: 82.7908 Compound 118

  Potency: 11.2202 Efficacy: 127.904 Compound 119

  Potency: 10 Efficacy: 114.827 Compound 120

  Potency: 8.9125 Efficacy: 92.062 Compound 121

  Potency: 10 Efficacy: 127.132 Compound 122

  Potency: 8.9125 Efficacy: 92.918 Compound 123

  Potency: 8.9125 Efficacy: 123.416 Compound 124

  Potency: 11.2202 Efficacy: 136.36 Compound 125

  Potency: 8.9125 Efficacy: 124.439 Compound 126

  Potency: 12.5893 Efficacy: 171.242 Compound 127

  Potency: 14.1254 Efficacy: 138.869 Compound 128

 

  Potency: 11.2202 Efficacy: 145.936 Compound 129

  Potency: 11.2202 Efficacy: 119.808 Compound 130

  Potency: 14.1254 Efficacy: 175.002 Compound 131

  Potency: 12.5893 Efficacy: 120.609 Compound 132

  Potency: 10 Efficacy: 100.712 Compound 133

  Potency: 10 Efficacy: 95.4818 Compound 134

  Potency: 10 Efficacy: 91.1222 Compound 135

  Potency: 8.9125 Efficacy: 89.1179 Compound 136

  Potency: 10 Efficacy: 81.023 Compound 137

  Potency: 11.2202 Efficacy: 135.363 Compound 138

  Potency: 11.2202 Efficacy: 67.3145 Compound 139

  Potency: 10 Efficacy: 81.9056 Compound 140

  Potency: 10 Efficacy: 100.669 Compound 141

  Potency: 8.9125 Efficacy: 89.9548 Compound 142

  Potency: 10 Efficacy: 76.5877 Compound 143

  Potency: 12.5893 Efficacy: 94.0383 Compound 144

  Potency: 10 Efficacy: 69.9943 Compound 145

  Potency: 10 Efficacy: 96.4095 Compound 146

  Potency: 11.2202 Efficacy: 90.1504 Compound 147

  Potency: 11.2202 Efficacy: 138.741 Compound 148

  Potency: 8.9125 Efficacy: 84.3701 Compound 149

  Potency: 10 Efficacy: 67.6116 Compound 150

  Potency: 10 Efficacy: 91.5246 Compound 151

  Potency: 12.5893 Efficacy: 97.2406 Compound 152

  Potency: 7.9433 Efficacy: 95.7207 Compound 153

  Potency: 3.1623 Efficacy: 93.8685 Compound 154

  Potency: 10 Efficacy: 93.0585 Compound 155

  Potency: 10 Efficacy: 67.8359 Compound 156

  Potency: 10 Efficacy: 70.1776 Compound 157

  Potency: 10 Efficacy: 71.256 Compound 158

  Potency: 11.2202 Efficacy: 109.576 Compound 159

  Potency: 11.2202 Efficacy: 103.065 Compound 160

  Potency: 3.9811 Efficacy: 97.9916 Compound 161

  Potency: 5.6234 Efficacy: 87.0645 Compound 162

  Potency: 10 Efficacy: 79.1483 Compound 163

  Potency: 10 Efficacy: 90.988 Compound 164

  Potency: 11.2202 Efficacy: 128.695 Compound 165

  Potency: 12.5893 Efficacy: 93.7937 Compound 166

  Potency: 7.0795 Efficacy: 86.8199 Compound 167

  Potency: 8.9125 Efficacy: 83.6415 Compound 168

  Potency: 10 Efficacy: 84.5587 Compound 169

  Potency: 10 Efficacy: 84.3344 Compound 170

  Potency: 10 Efficacy: 59.61 Compound 171

  Potency: 10 Efficacy: 65.8043 Compound 172

  Potency: 11.2202 Efficacy: 85.4536 Compound 173

  Potency: 11.2202 Efficacy: 104.045 Compound 174

  Potency: 5.6234 Efficacy: 116.69 Compound 175

  Potency: 10 Efficacy: 115.567 Compound 176

  Potency: 10 Efficacy: 70.6131 Compound 177

  Potency: 11.2202 Efficacy: 107.982 Compound 178

  Potency: 5.6234 Efficacy: 84.0774 Compound 179

  Potency: 10 Efficacy: 77.6845 Compound 180

  Potency: 12.5893 Efficacy: 80.5239 Compound 181

  Potency: 10 Efficacy: 99.1066 Compound 182

  Potency: 10 Efficacy: 98.6809 Compound 183

  Potency: 10 Efficacy: 86.1315 Compound 184

  Potency: 10 Efficacy: 117.72 Compound 185

  Potency: 3.9811 Efficacy: 93.5129 Compound 186

  Potency: 8.9125 Efficacy: 120.104 Compound 187

  Potency: 10 Efficacy: 88.0312 Compound 188

  Potency: 8.9125 Efficacy: 76.0346 Compound 189

  Potency: 11.2202 Efficacy: 116.431 Compound 190

  Potency: 11.2202 Efficacy: 115.598 Compound 191

  Potency: 11.2202 Efficacy: 74.0117 Compound 192

  Potency: 11.2202 Efficacy: 89.7146 Compound 193

  Potency: 8.9125 Efficacy: 76.8665 Compound 194

  Potency: 10 Efficacy: 99.4569 Compound 195

  Potency: 10 Efficacy: 67.0912 Compound 196

  Potency: 8.9125 Efficacy: 83.2038 Compound 197

  Potency: 10 Efficacy: 78.0861 Compound 198

  Potency: 12.5893 Efficacy: 96.0151 Compound 199

  Potency: 8.9125 Efficacy: 98.6316 Compound 200

  Potency: 11.2202 Efficacy: 94.7942 Compound 201

  Potency: 10 Efficacy: 83.9426 Compound 202

  Potency: 7.0795 Efficacy: 106.69 Compound 203

  Potency: 10 Efficacy: 70.2778 Compound 204

  Potency: 7.0795 Efficacy: 98.0553 Compound 205

  Potency: 11.2202 Efficacy: 77.9394 Compound 206

  Potency: 10 Efficacy: 75.3009 Compound 207

  Potency: 10 Efficacy: 83.7154 Compound 208

  Potency: 15.8489 Efficacy: 130.232 Compound 209

  Potency: 12.5893 Efficacy: 101.354 Compound 210

  Potency: 11.2202 Efficacy: 109.939 Compound 211

  Potency: 14.1254 Efficacy: 119.538 Compound 212

  Potency: 11.2202 Efficacy: 110.24 Compound 213

  Potency: 11.2202 Efficacy: 94.2928 Compound 214

  Potency: 10 Efficacy: 73.527 Compound 215

  Potency: 10 Efficacy: 90.7389 Compound 216

  Potency: 10 Efficacy: 56.3855 Compound 217

  Potency: 10 Efficacy: 58.0835 Compound 218

  Potency: 11.2202 Efficacy: 83.9475 Compound 219

  Potency: 11.2202 Efficacy: 86.9112 Compound 220

  Potency: 11.2202 Efficacy: 59.5046 Compound 221

  Potency: 12.5893 Efficacy: 56.9925 Compound 222

TABLE 8 Compound data Fit Fit Infinite Fit Zero Fit Curve Fit Hill Activity Activity Curve No. Score Description LogAC50 Slope Fit R2 [%] [%] Class 1 94 Complete −5.95 3.132 0.9985 195.171 −2.9752 1.1 curve; high efficacy 2 91 Complete −5.45 1.4163 0.9999 231.285 3.1995 1.1 curve; high efficacy 3 89 Complete −5.65 3.0654 0.9999 158.124 −35.3084 1.1 curve; high efficacy 4 87 Complete −5.55 1.8617 0.9998 136.905 7.6069 1.1 curve; high efficacy 5 86 Complete −5.95 2.5334 0.9999 87.4201 −5.0637 1.1 curve; high efficacy 6 85 Complete −5.95 1.3437 0.9999 69.589 −58.5311 1.1 curve; high efficacy 7 85 Complete −5.35 1.6924 0.9999 107.421 12.5 1.1 curve; high efficacy 8 85 Complete −5.55 4.9549 0.9996 96.0327 −15.6523 1.1 curve; high efficacy 9 85 Complete −5.55 1.3723 0.9999 92.2022 −30.5005 1.1 curve; high efficacy 10 85 Complete −5.5 2.8473 1 102.022 0 1.1 curve; high efficacy 11 85 Complete −5.4 2.5334 0.9997 101.384 −0.509 1.1 curve; high efficacy 12 85 Complete −5.55 1.4781 0.9999 102.305 9.3996 1.1 curve; high efficacy 13 84 Complete −5.35 3.9295 0.9991 94.7101 −27.2857 1.1 curve; high efficacy 14 84 Complete −5.65 2.3332 0.9995 62.8391 −21.9345 1.1 curve; high efficacy 15 84 Complete −5.5 1.6259 0.9999 75.2612 −16.1195 1.1 curve; high efficacy 16 84 Complete −5.65 1.3443 0.9999 77.492 −37.8568 1.1 curve; high efficacy 17 84 Complete −5.6 1.2475 0.9998 71.4307 −24.7641 1.1 curve; high efficacy 18 84 Partial −5 4.9549 0.9995 1760.36 8.7984 2.1 curve; high efficacy 19 84 Complete −5.85 1.3437 0.9992 52.7174 −28.8963 1.1 curve; high efficacy 20 84 Complete −5.45 2.3031 0.9999 83.8587 −17.7204 1.1 curve; high efficacy 21 84 Complete −5.25 4.4495 0.9992 93.4556 1.1731 1.1 curve; high efficacy 22 84 Complete −5.35 4.9549 0.9999 83.1353 −0.2899 1.1 curve; high efficacy 23 84 Complete −5.4 2.9023 0.9998 98.4745 13.3122 1.1 curve; high efficacy 24 84 Complete −5.5 2.1211 0.9996 82.7045 5.7274 1.1 curve; high efficacy 25 84 Complete −5.65 1.6604 0.9995 64.1141 −18.7854 1.1 curve; high efficacy 26 84 Complete −5.7 1.1 1 59.5556 −36.1807 1.1 curve; high efficacy 27 83 Complete −5.7 2.2526 0.9994 54.9511 −26.8995 1.1 curve; high efficacy 28 83 Complete −5.55 2.5334 0.9999 55.3257 −36.6914 1.1 curve; high efficacy 29 83 Complete −5.4 1.7885 0.9997 55.7271 −25.4387 1.1 curve; high efficacy 30 83 Complete −5.4 2.1876 0.9999 73.6408 −19.157 1.1 curve; high efficacy 31 83 Complete −5.6 1.9673 0.9999 62.1733 −24.2621 1.1 curve; high efficacy 32 83 Complete −5.55 2.1211 1 60.2337 −28.5623 1.1 curve; high efficacy 33 82 Complete −5.7 1.1 0.9991 33.1603 −50.1655 1.1 curve; high efficacy 34 65 Complete −5.85 1.8851 0.9995 76.3552 5.9015 1.2 curve; partial efficacy 35 64 Complete −5.6 3.132 0.9995 72.5763 −0.1751 1.2 curve; partial efficacy 36 64 Complete −5.5 1.4163 0.9997 81.2047 5.5 1.2 curve; partial efficacy 37 64 Complete −5.6 2.4064 0.9991 74.4523 −2.5935 1.2 curve; partial efficacy 38 63 Complete −5.35 4.9549 0.9992 73.5449 −2.0156 1.2 curve; partial efficacy 39 63 Complete −5.45 1.7529 0.9998 69.5378 −9.0026 1.2 curve; partial efficacy 40 48 Partial −4.85 4.5045 0.9986 433.479 −3.212 2.1 curve; high efficacy 41 48 Partial −4.85 4.5045 1 456.749 8.8874 2.1 curve; high efficacy 42 48 Partial −4.85 4.9549 0.9987 484.883 9.6207 2.1 curve; high efficacy 43 47 Partial −5.4 0.9 0.9999 148.01 0 2.1 curve; high efficacy 44 47 Partial −4.95 4.095 0.9997 310.669 −17.6073 2.1 curve; high efficacy 45 47 Partial −4.85 4.5045 0.9989 419.509 −7.0263 2.1 curve; high efficacy 46 46 Partial −5.1 1.01 0.9992 195.813 −35.6022 2.1 curve; high efficacy 47 46 Partial −5.25 1.331 0.9993 153.844 12.5272 2.1 curve; high efficacy 48 46 Partial −5.15 1.7137 0.9992 178.694 −35.4511 2.1 curve; high efficacy 49 46 Partial −5.25 1.9282 0.9996 153.971 2.973 2.1 curve; high efficacy 50 46 Partial −5.1 1.8851 0.9989 186.381 5.0579 2.1 curve; high efficacy 51 46 Partial −5 4.9549 0.9988 249.6 −23.8252 2.1 curve; high efficacy 52 46 Partial −5.35 1.4781 0.9998 137.586 −27.7073 2.1 curve; high efficacy 53 45 Partial −5.15 0.8 0.9991 152.283 −11.267 2.1 curve; high efficacy 54 45 Partial −4.9 1.1341 1 262.045 39.3368 2.1 curve; high efficacy 55 45 Partial −4.9 4.9549 0.9985 252.825 −18.4203 2.1 curve; high efficacy 56 45 Partial −5.4 1.5095 0.9999 110.65 21.8862 2.1 curve; high efficacy 57 45 Partial −5.35 1.7529 0.9999 115.173 −0.5596 2.1 curve; high efficacy 58 45 Partial −5.45 0.9 0.999 95.6282 −42.3624 2.1 curve; high efficacy 59 45 Partial −5 3.5722 0.9991 185.351 −3.6299 2.1 curve; high efficacy 60 45 Partial −5.35 1.7529 0.9992 116.389 −12.5146 2.1 curve; high efficacy 61 45 Partial −5.3 1.1705 0.9991 136.382 −3.94 2.1 curve; high efficacy 62 45 Partial −5.15 1.9673 0.9992 162.393 −23.864 2.1 curve; high efficacy 63 45 Partial −5 3.1925 0.9993 198.688 −1.7425 2.1 curve; high efficacy 64 45 Partial −5.3 2.4729 0.9996 129.946 −4.9734 2.1 curve; high efficacy 65 45 Partial −4.7 4.095 0.9991 501.215 9.2299 2.1 curve; high efficacy 66 45 Partial −4.9 4.5045 0.9991 227.168 11.9175 2.1 curve; high efficacy 67 44 Partial −5 1.4163 0.9999 162.351 4.7344 2.1 curve; high efficacy 68 44 Partial −4.9 4.5045 0.9986 221.625 −17.9914 2.1 curve; high efficacy 69 44 Partial −5.4 1.3987 0.9999 88.0721 −2.436 2.1 curve; high efficacy 70 44 Partial −5.35 1.2876 0.9999 96.1281 −19.1057 2.1 curve; high efficacy 71 44 Partial −5.1 1.3443 0.9999 122.331 −2.1806 2.1 curve; high efficacy 72 44 Partial −5.05 2.3332 0.9987 136.354 −2.3089 2.1 curve; high efficacy 73 44 Partial −4.9 4.095 0.9993 186.64 16.3113 2.1 curve; high efficacy 74 44 Partial −5.35 1.8617 1 86.169 −23.105 2.1 curve; high efficacy 75 44 Partial −5.4 1.5579 0.9999 98.3982 26.9551 2.2 curve; partial efficacy 76 44 Partial −5.2 1.3443 0.9997 105.907 −8.518 2.1 curve; high efficacy 77 44 Partial −5.35 1.8617 1 87.1172 −34.2328 2.1 curve; high efficacy 78 44 Partial −5 1.7137 0.9999 162.598 9.6566 2.1 curve; high efficacy 79 44 Partial −5.35 1.3437 0.9999 84.8433 −0.3646 2.1 curve; high efficacy 80 44 Partial −5 1.4163 0.9999 169.032 −6.484 2.1 curve; high efficacy 81 44 Partial −4.9 4.5045 0.9998 216.419 3.1931 2.1 curve; high efficacy 82 44 Partial −5.5 1.1341 1 89.9991 −6.4745 2.1 curve; high efficacy 83 44 Partial −4.95 2.0937 1 162.065 −12.9886 2.1 curve; high efficacy 84 44 Partial −5.2 1.2221 0.9999 114.278 −7.9639 2.1 curve; high efficacy 85 44 Partial −5.3 1.8617 0.9999 102.938 3.2331 2.1 curve; high efficacy 86 44 Partial −5.35 0.8 0.9999 94.6981 0.4963 2.1 curve; high efficacy 87 44 Partial −5.3 2.1211 0.9997 87.756 21.3502 2.2 curve; partial efficacy 88 43 Partial −5.05 1.01 0.9999 98.4045 9.418 2.1 curve; high efficacy 89 43 Partial −5.3 1.6924 0.999 75.3022 −10.8957 2.2 curve; partial efficacy 90 43 Partial −4.95 4.5045 0.999 146.447 5.7331 2.1 curve; high efficacy 91 43 Partial −4.95 4.095 0.9986 122.391 −16.7058 2.1 curve; high efficacy 92 43 Partial −5.35 1.6259 0.9999 76.9712 1.7602 2.2 curve; partial efficacy 93 43 Partial −4.95 1.7529 1 114.284 −3.6365 2.1 curve; high efficacy 94 43 Partial −5 4.9549 0.9996 138.031 9.6823 2.1 curve; high efficacy 95 43 Partial −5.15 1.01 0.9993 83.3763 −14.8966 2.2 curve; partial efficacy 96 43 Partial −5.35 1.9887 0.9998 74.9858 −8.0822 2.2 curve; partial efficacy 97 43 Partial −5.25 3.132 0.9994 79.8262 1.2108 2.2 curve; partial efficacy 98 43 Partial −5 4.5045 1 125.633 −0.0479 2.1 curve; high efficacy 99 43 Partial −5 2.1211 0.9999 121.073 0 2.1 curve; high efficacy 100 43 Partial −5.4 1.01 0.9992 64.0202 −31.5116 2.1 curve; high efficacy 101 43 Partial −5.05 1.6604 0.9989 115.659 −15.8708 2.1 curve; high efficacy 102 43 Partial −4.9 4.5045 0.9999 160.171 10.4042 2.1 curve; high efficacy 103 43 Partial −5.2 1.8265 0.9986 96.8922 −23.0045 2.1 curve; high efficacy 104 43 Partial −5 3.6772 0.9988 128.477 −9.1182 2.1 curve; high efficacy 105 43 Partial −5 3.5117 0.9995 113.218 16.8467 2.1 curve; high efficacy 106 43 Partial −5.15 1.21 1 79.2242 −2.155 2.2 curve; partial efficacy 107 43 Partial −5.35 1.4781 0.9998 76.3701 −19.2206 2.1 curve; high efficacy 108 43 Partial −4.9 4.5045 0.9999 142.05 2.0834 2.1 curve; high efficacy 109 43 Partial −4.9 4.9549 0.9994 174.079 7.5839 2.1 curve; high efficacy 110 43 Partial −4.9 4.5045 0.9999 162.565 −18.2174 2.1 curve; high efficacy 111 43 Partial −5.15 1.21 0.9999 101.431 2.2101 2.1 curve; high efficacy 112 43 Partial −5.25 3.132 1 74.0416 0.1229 2.2 curve; partial efficacy 113 43 Partial −5 3.9295 0.9994 105.867 −5.8907 2.1 curve; high efficacy 114 43 Partial −4.95 4.5045 0.9989 134.828 −3.8223 2.1 curve; high efficacy 115 43 Partial −5.15 1.8265 0.9997 80.6607 −4.8829 2.2 curve; partial efficacy 116 43 Partial −5.05 1.5579 0.9999 94.7519 −9.8246 2.1 curve; high efficacy 117 43 Partial −5.1 1.6604 0.9998 88.7778 −0.2202 2.1 curve; high efficacy 118 43 Partial −5.3 1.6924 0.9992 74.8733 −7.9176 2.2 curve; partial efficacy 119 43 Partial −4.95 4.9549 0.9997 111.883 −16.0213 2.1 curve; high efficacy 120 43 Partial −5 0.8 0.9996 126.76 11.933 2.1 curve; high efficacy 121 43 Partial −5.05 1.8851 0.9997 97.7753 5.7133 2.1 curve; high efficacy 122 43 Partial −5 1.2475 0.9999 127.656 0.5248 2.1 curve; high efficacy 123 43 Partial −5.05 1.3443 0.9999 96.9016 3.9836 2.1 curve; high efficacy 124 43 Partial −5.05 1.2475 0.9999 102.977 −20.4393 2.1 curve; high efficacy 125 43 Partial −4.95 1.1705 0.9999 133.669 −2.6906 2.1 curve; high efficacy 126 43 Partial −5.05 1 1 125.112 0.673 2.1 curve; high efficacy 127 43 Partial −4.9 4.5045 0.9995 152.332 −18.9102 2.1 curve; high efficacy 128 43 Partial −4.85 4.5045 0.9991 153.542 14.6735 2.1 curve; high efficacy 129 43 Partial −4.95 4.095 0.9985 128.069 −17.8675 2.1 curve; high efficacy 130 43 Partial −4.95 4.5045 0.9998 127.62 7.8122 2.1 curve; high efficacy 131 43 Partial −4.85 4.9549 0.9997 183.761 8.7591 2.1 curve; high efficacy 132 43 Partial −4.9 4.9549 0.9996 133.42 12.8108 2.1 curve; high efficacy 133 42 Partial −5 2.7202 0.9992 73.1459 −27.5656 2.1 curve; high efficacy 134 42 Partial −5 4.4495 0.9993 72.9186 −22.5632 2.1 curve; high efficacy 135 42 Partial −5 4.9549 0.9997 61.8199 −29.3022 2.1 curve; high efficacy 136 42 Partial −5.05 0.8 0.9997 85.8101 −3.3079 2.2 curve; partial efficacy 137 42 Partial −5 4.9549 0.9999 61.7369 −19.2861 2.1 curve; high efficacy 138 42 Partial −4.95 1.3437 1 107.998 −27.3652 2.1 curve; high efficacy 139 42 Partial −4.95 4.5045 0.9999 82.0264 14.712 2.1 curve; high efficacy 140 42 Partial −5 2.7868 0.9991 77.8665 −4.0391 2.2 curve; partial efficacy 141 42 Partial −5 4.9549 0.9998 82.7722 −17.8968 2.1 curve; high efficacy 142 42 Partial −5.05 2.4064 0.9988 71.1773 −18.7775 2.1 curve; high efficacy 143 42 Partial −5 4.4495 0.9999 81.2596 4.6719 2.1 curve; high efficacy 144 42 Partial −4.9 4.095 0.9995 103.816 9.7776 2.1 curve; high efficacy 145 42 Partial −5 4.9549 0.9999 70.4611 0.4668 2.2 curve; partial efficacy 146 42 Partial −5 4.9549 0.9999 75.4854 −20.924 2.1 curve; high efficacy 147 42 Partial −4.95 2.0937 0.9999 91.2034 1.0531 2.1 curve; high efficacy 148 42 Partial −4.95 2.3531 0.9997 109.969 −28.7719 2.1 curve; high efficacy 149 42 Partial −5.05 1.111 0.9999 82.4023 −1.9678 2.2 curve; partial efficacy 150 42 Partial −5 4.5045 0.9999 67.4005 −0.2112 2.2 curve; partial efficacy 151 42 Partial −5 3.132 0.9999 65.1432 −26.3814 2.1 curve; high efficacy 152 42 Partial −4.9 1.1341 0.9999 88.7605 −8.4801 2.2 curve; partial efficacy 153 42 Partial −5.1 1.5095 0.9998 74.4536 −21.2671 2.1 curve; high efficacy 154 42 Partial −5.5 0.9 0.9999 47.5155 −46.353 2.1 curve; high efficacy 155 42 Partial −5 3.99 0.9998 60.936 −32.1225 2.1 curve; high efficacy 156 42 Partial −5 2.5334 0.9999 67.8023 −0.0336 2.2 curve; partial efficacy 157 42 Partial −5 3.6272 0.9989 68.2216 −1.9559 2.2 curve; partial efficacy 158 42 Partial −5 1.9282 0.9994 70.7327 −0.5234 2.2 curve; partial efficacy 159 42 Partial −4.95 4.095 0.9995 106.672 −2.9033 2.1 curve; high efficacy 160 42 Partial −4.95 4.095 0.9998 100.584 −2.4804 2.1 curve; high efficacy 161 42 Partial −5.4 1.1 0.9998 55.0498 −42.9418 2.1 curve; high efficacy 162 42 Partial −5.25 2.2526 1 55.2524 −31.8121 2.1 curve; high efficacy 163 42 Partial −5 4.045 0.9999 78.2931 −0.8551 2.2 curve; partial efficacy 164 42 Partial −5 4.5045 0.9998 91.825 0.8371 2.1 curve; high efficacy 165 42 Partial −4.95 4.5045 0.9999 108.575 −20.12 2.1 curve; high efficacy 166 42 Partial −4.9 1.3987 0.9998 104.163 10.3691 2.1 curve; high efficacy 167 42 Partial −5.15 1.7885 0.9995 48.5966 −38.2233 2.1 curve; high efficacy 168 42 Partial −5.05 1.9673 0.9989 82.861 −0.7805 2.2 curve; partial efficacy 169 42 Partial −5 4.4495 0.9987 64.8483 −19.7104 2.1 curve; high efficacy 170 42 Partial −5 4.4495 0.9988 87.8075 3.4732 2.1 curve; high efficacy 171 42 Partial −5 3.132 0.9999 69.5258 9.9159 2.2 curve; partial efficacy 172 42 Partial −5 2.3332 0.9987 73.8043 8 2.2 curve; partial efficacy 173 42 Partial −4.95 0.9 0.9987 93.0488 7.5952 2.2 curve; partial efficacy 174 42 Partial −4.95 1.8579 0.9993 83.6585 −20.386 2.1 curve; high efficacy 175 42 Partial −5.25 1.3443 0.9985 54.0029 −62.687 2.1 curve; high efficacy 176 42 Partial −5 4.9549 0.9985 96.131 −19.4361 2.1 curve; high efficacy 177 42 Partial −5 4.9549 0.9998 71.5284 0.9153 2.2 curve; partial efficacy 178 42 Partial −4.95 1.1341 0.9994 85.3274 −22.6542 2.1 curve; high efficacy 179 42 Partial −5.25 2.2526 0.9998 48.386 −35.6913 2.1 curve; high efficacy 180 42 Partial −5 3.1925 0.9991 76.0607 −1.6238 2.2 curve; partial efficacy 181 42 Partial −4.9 4.9549 1 93.0239 12.5 2.1 curve; high efficacy 182 42 Partial −5 2.2481 0.9998 98.2495 −0.8571 2.1 curve; high efficacy 183 42 Partial −5 4.4495 0.9997 99.728 1.0471 2.1 curve; high efficacy 184 42 Partial −5 2.9023 0.9998 85.5304 −0.6011 2.1 curve; high efficacy 185 42 Partial −5 4.9549 0.9994 94.2492 −23.4705 2.1 curve; high efficacy 186 42 Partial −5.4 0.9 0.9999 47.9827 −45.5302 2.1 curve; high efficacy 187 42 Partial −5.05 1.3437 0.9992 65.7373 −54.3669 2.1 curve; high efficacy 188 42 Partial −5 4.5045 0.9999 88.2262 0.195 2.1 curve; high efficacy 189 42 Partial −5.05 0.8 0.9999 84.5737 8.5392 2.2 curve; partial efficacy 190 42 Partial −4.95 2.0937 0.9991 87.524 −28.9068 2.1 curve; high efficacy 191 42 Partial −4.95 4.9549 0.9998 96.2795 −19.3188 2.1 curve; high efficacy 192 42 Partial −4.95 4.095 0.9992 76.4906 2.4789 2.2 curve; partial efficacy 193 42 Partial −4.95 4.095 0.9997 92.2616 2.547 2.1 curve; high efficacy 194 42 Partial −5.05 1.7137 0.9999 73.9084 −2.9581 2.2 curve; partial efficacy 195 42 Partial −5 4.045 0.9987 89.5777 −9.8792 2.1 curve; high efficacy 196 42 Partial −5 2.8473 0.9999 66.5821 −0.5091 2.2 curve; partial efficacy 197 42 Partial −5.05 0.9 0.9999 84.7038 1.5 2.2 curve; partial efficacy 198 42 Partial −5 4.9549 0.9993 79.1775 1.0914 2.2 curve; partial efficacy 199 42 Partial −4.9 1.2876 0.9988 96.2012 0.1862 2.2 curve; partial efficacy 200 42 Partial −5.05 1.4781 0.9999 56.5654 −42.0662 2.1 curve; high efficacy 201 42 Partial −4.95 2.3531 0.9987 74.9126 −19.8816 2.1 curve; high efficacy 202 42 Partial −5 1.8617 0.9999 80.5565 −3.3861 2.2 curve; partial efficacy 203 42 Partial −5.15 1.4781 0.9998 59.3901 −47.2995 2.1 curve; high efficacy 204 42 Partial −5 3.5722 0.9999 70.7642 0.4864 2.2 curve; partial efficacy 205 42 Partial −5.15 1.4781 0.9999 57.7575 −40.2978 2.1 curve; high efficacy 206 42 Partial −4.95 4.095 0.9985 91.8921 13.9528 2.1 curve; high efficacy 207 42 Partial −5 2.5334 0.9998 75.4708 0.1699 2.2 curve; partial efficacy 208 42 Partial −5 3.1925 0.9988 74.5741 −9.1413 2.2 curve; partial efficacy 209 42 Partial −4.8 4.095 0.9997 142.41 12.1779 2.1 curve; high efficacy 210 42 Partial −4.9 4.9549 0.9986 113.27 11.9156 2.1 curve; high efficacy 211 42 Partial −4.95 1.1341 0.9991 109.674 −0.265 2.1 curve; high efficacy 212 42 Partial −4.85 4.5045 0.9993 128.55 9.0119 2.1 curve; high efficacy 213 42 Partial −4.95 1.2876 0.9999 109.228 −1.0122 2.1 curve; high efficacy 214 42 Partial −4.95 2.0937 0.9997 108.196 13.9034 2.1 curve; high efficacy 215 42 Partial −5 2.1211 0.9999 87.4293 13.9023 2.1 curve; high efficacy 216 42 Partial −5 0.8 0.9996 94.4089 3.67 2.2 curve; partial efficacy 217 42 Partial −5 3.0654 0.9995 66.1813 9.7958 2.2 curve; partial efficacy 218 42 Partial −5 3.6272 0.9998 71.8402 13.7566 2.2 curve; partial efficacy 219 42 Partial −4.95 1.5936 0.9993 76.3682 −7.5793 2.2 curve; partial efficacy 220 42 Partial −4.95 3.2975 0.9995 95.08 8.1688 2.1 curve; high efficacy 221 41 Partial −4.95 4.095 0.9994 66.5046 7 2.2 curve; partial efficacy 222 41 Partial −4.9 4.095 0.9999 66.8444 9.8519 2.2 curve; partial efficacy Activity Activity Activity at Activity at at Max 0.307 at 1.530 7.660 38.30 Excl'd Response uM uM uM uM No. Points [%] [%]** [%]** [%]** [%]**  1 0 0 0 0 190.551 0 142.678 198.344 190.551  2 0 0 0 0 222.818 9.5199 56.9884 175.022 222.818  3 0 0 0 0 158.59 −34.2446 9.5695 153.966 158.59  4 0 0 0 0 136.632 9.8511 38.314 118.549 136.632  5 0 0 0 0 87.1241 −1.7993 58.4455 87.2456 87.1241  6 0 0 0 0 68.1048 −39.7851 19.1903 60.5602 68.1048  7 0 0 0 0 104.699 13.4132 25.2727 80.6487 104.699  8 0 0 0 0 94.3383 −15.3725 −10.6082 96.4184 94.3383  9 0 0 0 0 88.6572 −24.4743 6.2893 68.0567 88.6572  10 0 0 0 0 101.819 0 11.3964 94.1328 101.819  11 0 0 0 0 101.791 0 7.5981 83.8704 101.791  12 0 0 0 0 100.299 12.3078 36.4535 84.576 100.299  13 0 0 0 0 95.6495 −24.7665 −27.3506 80.6874 95.6495  14 0 0 0 0 61.7295 −21.6121 3.5416 59.0823 61.7295  15 0 0 0 0 73.6236 −13.6594 4.7051 58.0277 73.6236  16 0 0 0 0 75.2018 −30.2295 5.6414 58.1985 75.2018  17 0 0 0 0 67.6004 −18.3073 9.3675 52.6808 67.6004  18 0 0 0 0 1767.43 7.745 30.584 357.845 1767.43  19 0 0 0 0 52.0677 −20.1458 14.9627 43.8719 52.0677  20 0 0 0 0 82.8998 −18.1348 −4.4619 69.3278 82.8998  21 0 0 0 0 93.2691 2.856 0 75.5731 93.2691  22 0 0 0 0 82.8991 0 0 78.2915 82.8991  23 0 0 0 0 98.87 12.6255 18.8414 87.3421 98.87  24 0 0 0 0 81.3384 5.8865 19.6198 73.0848 81.3384  25 0 0 0 0 63.0097 −16.5079 11.1354 54.5336 63.0097  26 0 0 0 0 56.0664 −25.4416 4.5885 41.9493 56.0664  27 0 0 0 0 53.7617 −26.1631 3.3138 51.6127 53.7617  28 0 0 0 0 54.6586 −35.9928 −20.5758 48.8507 54.6586  29 0 0 0 0 54.9055 −23.8063 −13.4709 35.9591 54.9055  30 0 0 0 0 73.4371 −18.3179 −9.0684 55.4601 73.4371  31 0 0 0 0 62.3312 −22.9164 −0.943 53.2493 62.3312  32 0 0 0 0 59.833 −28.0565 −9.4589 51.0458 59.833  33 0 0 0 0 31.0963 −41.8046 −14.3237 16.9691 31.0963  34 0 0 0 0 75.3947 9.4093 43.8029 74.3361 75.3947  35 0 0 0 0 71.7515 0 12.0467 71.5303 71.7515  36 0 0 0 0 78.5607 7.6747 25.7129 65.0516 78.5607  37 0 0 0 0 72.867 −1.7745 15.279 70.7278 72.867  38 0 0 0 0 72.6726 −0.6248 −2.8833 69.1705 72.6726  39 0 0 0 0 69.0926 −7.7641 5.5069 52.9348 69.0926  40 0 0 0 0 427.494 −13.7066 5.577 25.2218 427.494  41 0 0 0 0 453.124 8.7547 9.7092 34.1487 453.124  42 0 0 0 0 481.034 20.1308 0 32.0577 481.034  43 0 0 0 0 131.214 13.6807 43.9438 94.2627 131.214  44 0 0 0 0 308.204 −13.4703 −18.7598 38.1168 308.204  45 0 0 0 0 413.716 −16.6135 0.0514 18.6133 413.716  46 0 0 0 0 156.151 −29.6685 4.4407 77.5979 156.151  47 0 0 0 0 142.448 17.1767 32.1719 97.3497 142.448  48 0 0 0 0 165.458 −32.0109 −23.2923 76.5902 165.458  49 0 0 0 0 150.952 2.3042 15.4629 102.178 150.952  50 0 0 0 0 176.497 7.9011 9.4014 94.1426 176.497  51 0 0 0 0 249.102 −27.9345 −17.6035 31.4284 249.102  52 0 0 0 0 130.29 −25.6891 1.731 86.3564 130.29  53 0 0 0 0 118.6 0 28.0603 71.9463 118.6  54 0 0 0 0 213.045 42.0153 58.6176 119.863 213.045  55 0 0 0 0 252.32 −24.5803 −12.1219 3.459 252.32  56 0 0 0 0 107.846 23.9844 38.0808 86.6615 107.846  57 0 0 0 0 112.914 1.0034 14.5781 82.1622 112.914  58 0 0 0 0 82.8269 −29.6769 3.3459 49.0869 82.8269  59 0 0 0 0 183.88 0 −6.1928 48.4893 183.88  60 0 0 0 0 115.466 −10.9556 4.4512 78.2781 115.466  61 0 0 0 0 126.28 −0.1233 25.8337 82.3962 126.28  62 0 0 0 0 154.66 −21.3534 −18.0137 77.9021 154.66  63 0 0 0 0 195.944 −3.9508 2.2361 57.8488 195.944  64 0 0 0 0 128.914 −6.3997 3.462 95.0937 128.914  65 0 0 0 0 464.087 8.214 2.5941 27.1667 464.087  66 0 0 0 0 225.365 16.1816 8.6821 32.7652 225.365  67 0 0 0 0 141.667 6.398 14.4975 69.8045 141.667  68 0 0 0 0 219.866 −11.823 −22.0487 4.4563 219.866  69 0 0 0 0 84.3602 0 16.1931 62.7677 84.3602  70 0 0 0 0 89.5048 −15.4839 4.1098 57.1687 89.5048  71 0 0 0 0 109.03 0 9.6296 58.7582 109.03  72 0 0 0 0 132.126 0.9098 −2.4849 54.5396 132.126  73 0 0 0 0 185.158 13.4604 18.6219 35.6621 185.158  74 0 0 0 0 84.2199 −22.8223 −9.7273 56.677 84.2199  75 0 0 0 0 95.9047 28.0842 39.9836 79.7093 95.9047  76 0 0 0 0 96.9841 −5.8134 5.6525 55.1076 96.9841  77 0 0 0 0 85.0608 −33.466 −19.7149 54.711 85.0608  78 0 0 0 0 148.085 10.1341 15.4808 69.8478 148.085  79 0 0 0 0 80.1706 1.5242 16.235 57.3461 80.1706  80 0 0 0 0 145.969 −4.7753 4.5639 65.9307 145.969  81 0 0 0 0 214.702 1.8272 5.1171 24.2861 214.702  82 0 0 0 0 84.7449 0 23.1094 63.7317 84.7449  83 0 0 0 0 149.231 −13.5366 −10.5766 41.5268 149.231  84 0 0 0 0 102.4 −5.2905 11.0012 59.7194 102.4  85 0 0 0 0 100.92 3.2508 13.2564 71.6469 100.92  86 0 0 0 0 80.4147 10.3775 28.9147 57.3965 80.4147  87 0 0 0 0 86.5444 20.8761 26.4772 69.1414 86.5444  88 0 0 0 0 81.7691 12.5888 21.7607 50.5291 81.7691  89 0 0 0 0 74.1053 −9.9886 −0.2967 45.5813 74.1053  90 0 0 0 0 146.154 2.8256 8.1166 27.152 146.154  91 0 0 0 0 121.42 −19.1946 −13.3374 6.5712 121.42  92 0 0 0 0 74.9657 2.5679 13.3008 54.89 74.9657  93 0 0 0 0 101.857 −3.4693 0 36.266 101.857  94 0 0 0 0 137.756 11.4997 8.6578 36.5266 137.756  95 0 0 0 0 67.941 −12.4138 3.0575 36.2186 67.941  96 0 0 0 0 73.1974 −7.8942 1.1034 54.4732 73.1974  97 0 0 0 0 79.2643 1.9074 1.3725 59.0705 79.2643  98 0 0 0 0 125.383 −0.0399 0.1051 28.903 125.383  99 0 0 0 0 114.004 0.7442 1.7316 44.4339 114.004 100 0 0 0 0 56.3462 −24.5207 −4.9865 30.1418 56.3462 101 0 0 0 0 104.197 −14.2124 −11.5581 43.2955 104.197 102 0 0 0 0 158.9 10.2684 11.2981 24.3058 158.9 103 0 0 0 0 91.3206 −22.0036 −16.3773 50.2037 91.3206 104 0 0 0 0 127.457 −6.4868 −11.6559 27.3282 127.457 105 0 0 0 0 112.32 15.3847 17.8982 44.2423 112.32 106 0 0 0 0 70.115 −0.495 9.0228 40.4026 70.115 107 0 0 0 0 73.3496 −17.2197 −3.2434 45.9779 73.3496 108 0 0 0 0 140.923 2.0308 3.1422 15.3462 140.923 109 0 0 0 0 173.731 5.1999 10.0589 20.86 173.731 110 0 0 0 0 161.275 −18.9356 −17.8813 0 161.275 111 0 0 0 0 89.9207 4.1224 15.4494 54.5855 89.9207 112 0 0 0 0 73.7713 0 1.5319 54.0265 73.7713 113 0 0 0 0 105.027 −4.9426 −7.7278 23.4771 105.027 114 0 0 0 0 134.559 −6.7171 −1.2468 17.1799 134.559 115 0 0 0 0 77.6085 −3.9401 0 40.2449 77.6085 116 0 0 0 0 84.9032 −9.1033 −3.9425 36.7288 84.9032 117 0 0 0 0 82.2017 0 5.0693 43.6203 82.2017 118 0 0 0 0 73.5555 −8.1247 2.294 48.7391 73.5555 119 0 0 0 0 111.66 −17.1068 −14.6664 0.6285 111.66 120 0 0 0 0 97.3577 17.804 33.8372 63.3531 97.3577 121 0 0 0 0 92.5902 6.8559 8.4589 45.0425 92.5902 122 0 0 0 0 107.455 2.4969 11.4724 54.0343 107.455 123 0 0 0 0 85.4512 5.1227 12.1485 45.1238 85.4512 124 0 0 0 0 85.8569 −19.2219 −7.3929 35.4594 85.8569 125 0 0 0 0 107.624 0 9.2558 50.3725 107.624 126 0 0 0 0 101.717 5.1764 18.9708 58.0571 101.717 127 0 0 0 0 151.123 −20.0375 −16.1406 −2.9507 151.123 128 0 0 0 0 152.324 17.3412 12.5761 22.6146 152.324 129 0 0 0 0 127.052 −21.7078 −15.591 8.9584 127.052 130 0 0 0 0 127.365 7.1779 9.1804 26.1033 127.365 131 0 0 0 0 182.302 10.6925 7.1202 16.169 182.302 132 0 0 0 0 133.154 11.1461 13.8268 23.024 133.154 133 0 0 0 0 70.2432 −25.994 −28.4428 5.985 70.2432 134 0 0 0 0 72.8618 −20.9617 −23.9361 −0.0686 72.8618 135 0 0 0 0 62.005 −28.5367 −29.3616 −11.1775 62.005 136 0 0 0 0 65.1019 2.0231 14.5105 38.0502 65.1019 137 0 0 0 0 61.7759 −19.2435 −19.3578 −1.6699 61.7759 138 0 0 0 0 85.8466 −26.0685 −19.0518 23.5861 85.8466 139 0 0 0 0 81.696 15.171 14.5765 24.988 81.696 140 0 0 0 0 75.9628 −2.7644 −4.956 22.8114 75.9628 141 0 0 0 0 82.663 −17.1706 −18.2787 2.6033 82.663 142 0 0 0 0 68.9039 −17.1048 −18.6659 16.7527 68.9039 143 0 0 0 0 80.844 4.2822 4.8849 22.4204 80.844 144 0 0 0 0 102.992 8.5445 11.1102 20.4218 102.992 145 0 0 0 0 70.5123 0.3023 0.1807 15.6647 70.5123 146 0 0 0 0 75.4881 −20.8468 −20.9197 0 75.4881 147 0 0 0 0 84.9194 0.4502 2.7027 29.0697 84.9194 148 0 0 0 0 102.444 −30.0492 −26.8413 12.5122 102.444 149 0 0 0 0 68.0415 −0.1259 8.5917 37.0328 68.0415 150 0 0 0 0 67.1116 0 0.1599 15.1419 67.1116 151 0 0 0 0 63.6617 −26.5586 −25.9538 2.045 63.6617 152 0 0 0 0 67.3709 −6.7254 0 26.2892 67.3709 153 0 0 0 0 66.0451 −20.7719 −14.2846 26.1958 66.0451 154 0 0 0 0 38.9061 −36.1249 −14.4105 17.8641 38.9061 155 0 0 0 0 60.5343 −32.0766 −31.7653 −9.1234 60.5343 156 0 0 0 0 65.3359 0 0 22.9027 65.3359 157 0 0 0 0 67.4685 −0.7059 −3.266 17.7416 67.4685 158 0 0 0 0 65.8674 0.6705 0.6338 26.3415 65.8674 159 0 0 0 0 105.826 −4.2541 −1.9112 17.1057 105.826 160 0 0 0 0 99.7859 −2.4977 −3.3563 16.0042 99.7859 161 0 0 0 0 48.073 −37.0265 −17.738 22.4249 48.073 162 0 0 0 0 54.2827 −31.8337 −27.5288 26.3243 54.2827 163 0 0 0 0 78.1035 −0.8037 −0.3894 19.4261 78.1035 164 0 0 0 0 91.6418 1.1551 0.0123 22.043 91.6418 165 0 0 0 0 107.713 −20.5064 −20.4325 0 107.713 166 0 0 0 0 87.6791 10.2974 15.5578 41.6567 87.6791 167 0 0 0 0 43.999 −38.1941 −33.4689 9.5564 43.999 168 0 0 0 0 77.8049 0 35.7996 77.8049 169 0 0 0 0 64.3252 −18.1947 −21.5953 0.301 64.3252 170 0 0 0 0 87.6323 2.3641 5.3324 22.1704 87.6323 171 0 0 0 0 68.362 10.0449 10.2067 28.1342 68.362 172 0 0 0 0 71.1503 8.9457 7.2239 31.1775 71.1503 173 0 0 0 0 72.2674 10.3627 21.0562 42.1173 72.2674 174 0 0 0 0 74.1998 −21.7408 −16.656 13.5733 74.1998 175 0 0 0 0 48.1163 −59.8895 −45.4644 5.3909 48.1163 176 0 0 0 0 95.8578 −22.0943 −16.8813 5.2636 95.8578 177 0 0 0 0 71.1427 1.3136 0.2953 16.2008 71.1427 178 0 0 0 0 64.2561 −21.1531 −11.2645 18.8833 64.2561 179 0 0 0 0 46.8236 −35.3915 −32.175 20.8832 46.8236 180 0 0 0 0 75.0675 0 −2.3977 21.2822 75.0675 181 0 0 0 0 92.8383 12.3946 12.9169 18.8847 92.8383 182 0 0 0 0 93.5709 0 0 34.7191 93.5709 183 0 0 0 0 99.5289 0 2.0346 24.4291 99.5289 184 0 0 0 0 83.8533 0 −0.5506 26.0285 83.8533 185 0 0 0 0 94.548 −21.6443 −24.0045 0 94.548 186 0 0 0 0 37.2481 −37.158 −17.69 15.0968 37.2481 187 0 0 0 0 51.5472 −53.6342 −42.397 −1.7858 51.5472 188 0 0 0 0 88.0501 0 0.7946 19.9555 88.0501 189 0 0 0 0 66.7349 13.1405 23.7073 43.9763 66.7349 190 0 0 0 0 79.582 −30.5111 −25.2626 6.3098 79.582 191 0 0 0 0 96.0338 −18.0752 −19.9657 −4.197 96.0338 192 0 0 0 0 76.1007 0.8898 2.9636 15.9079 76.1007 193 0 0 0 0 91.5294 3.7365 1.9996 17.8759 91.5294 194 0 0 0 0 68.0475 −2.4257 0 30.6498 68.0475 195 0 0 0 0 89.3989 −7.8811 −12.0365 15.2586 89.3989 196 0 0 0 0 65.0912 0 0 20.6484 65.0912 197 0 0 0 0 66.8554 5.2461 15.7031 40.796 66.8554 198 0 0 0 0 79.0861 2.3908 0 17.574 79.0861 199 0 0 0 0 78.1385 0 7.6087 32.1891 78.1385 200 0 0 0 0 46.2423 −40.8885 −35.7652 1.5723 46.2423 201 0 0 0 0 69.7901 −21.5136 −17.1084 8.0866 69.7901 202 0 0 0 0 74.538 −3.4095 −0.6635 28.063 74.538 203 0 0 0 0 51.7897 −45.9905 −36.9465 8.4225 51.7897 204 0 0 0 0 70.2936 0.8815 0.1572 20.0962 70.2936 205 0 0 0 0 50.1627 −39.3964 −31.3886 12.0722 50.1627 206 0 0 0 0 91.1628 12.1077 15.2718 28.5203 91.1628 207 0 0 0 0 73.3009 0.545 0 25.4562 73.3009 208 0 0 0 0 73.4157 −7.8355 −10.7764 16.4066 73.4157 209 0 0 0 0 138.801 12.9601 10.613 18.5675 138.801 210 0 0 0 0 113.044 13.8203 9.4299 20.2893 113.044 211 0 0 0 0 87.88 0.2296 11.6146 43.0472 87.88 212 0 0 0 0 127.53 11.1275 7.371 15.9241 127.53 213 0 0 0 0 90.1221 0 6.4924 41.084 90.1221 214 0 0 0 0 101.307 13.239 16.0649 43.8718 101.307 215 0 0 0 0 83.2597 14.2367 15.1789 40.8331 83.2597 216 0 0 0 0 71.6749 8.2756 20.8299 43.9905 71.6749 217 0 0 0 0 65.2014 10.407 9.2853 27.4661 65.2014 218 0 0 0 0 71.4891 13.9594 13.1115 29.7899 71.4891 219 0 0 0 0 66.0085 −8.2231 −3.0469 21.4195 66.0085 220 0 0 0 0 93.7673 7.1434 9.5135 27.6356 93.7673 221 0 0 0 0 66.1162 6.3785 7.8673 16.8498 66.1162 222 0 0 0 0 66.4815 9.4439 9.7686 16.5469 66.4815

Example 3

In this example, embodiments of small molecules that increase α7 integrin in skeletal muscle are disclosed. Using myogenic cells from mice in which the LacZ reporter gene was inserted into exon 1 of the mouse α7 integrin gene, 403,000 compounds were screened and more than 1500 hits that increased the β-galactosidase reporter were identified. Further evaluation identified 6 compounds that increased α7 integrin at least 1.5-fold in myotubes. Compounds were classified as iron chelating compounds, cell cycle inhibitors and compounds with undefined function. Compounds identified from this screen represent novel molecular probes that can be used to further elucidate regulation of α7β1 integrin expression and signaling in skeletal muscle and may serve as potential therapeutics for the treatment of DMD.

Several therapeutic approaches have been developed with the aim of restoring dystrophin expression and shown efficacy in animal models of DMD. These include virally mediated delivery and expression of dystrophin, myoblast cell transfer and engraftment, exon-skipping and stop-codon read-through. Currently, none of these methods have been approved as therapy for DMD patients. An alternative approach is to target and enhance levels of proteins which modify disease progression and act to partially compensate for the absence of dystrophin. These disease modifiers include utrophin, IGF-1, α7β1 integrin, GalNac, nNos and Adam12.

The α7β1 integrin is a laminin receptor in skeletal muscle that serves to link laminin-211/221 in the basal lamina and the actin cytoskeleton of muscle. The α7β1 integrin has structural and signaling functions that contribute to muscle development and physiology and was originally identified as a marker for muscle differentiation. Studies have shown that enhanced transgenic expression of the α7 integrin in skeletal and cardiac muscle can ameliorate dystrophic pathology and extend the lifespan of mdx/utr^(−/−) mice more than three-fold. Multiple mechanisms appear to contribute to α7 integrin mediated rescue of dystrophin deficient muscle including maintenance of myotendinous and neuromuscular junctions, enhanced muscle hypertrophy and regeneration, and decreased apoptosis and cardiomyopathy. Enhanced α7 integrin also protects muscles against exercise-induced damage. Conversely loss of the α7 integrin in mdx mice results in more severe muscle disease. Together these observations support the idea that the α7β1 integrin is a major disease modifier in DMD.

To translate transgenic mouse studies into potential therapies for DMD, a drug discovery program to identify chemical probes that increase α7 integrin in skeletal muscle was initiated. A report on the generation and characterization of an α7 integrin knockout line of mice in which the LacZ gene is inserted into intron 1, downstream of the endogenous α7 integrin promoter has been reported. Thus, β-galactosidase functions as a reporter for α7 integrin expression in these animals. Primary myogenic cells were isolated from a heterozygous mouse (α7βgal^(+/−)) so that the cells express α7 integrin and also report for transcription of the integrin. The myogenic reporter cells were designated α7βgal^(+/−) and were used to identify two molecules, valproic acid and laminin-111, in preliminary screens and have been successfully tested in mouse models of DMD.

To identify further small molecules that increase α7 integrin in skeletal muscle, a muscle cell-based assay to screen 403,000 compounds including FDA approved drugs and the large compound libraries at the National Chemical and Genomics Center (NCGC) was used. Several compounds that increase the α7 integrin with known mechanisms of action including iron chelators, microtubule inhibitors, cell cycle inhibitors and steroid-like molecules were identified. Additionally, several small molecules with unknown biological activities were identified. Overall, the results identified novel small molecules that increase the α7 integrin in cultured muscle cells and may serve as molecular probes to further dissect signaling pathways that regulate the α7β1 integrin in skeletal muscle. These small molecules could potentially be developed as novel therapeutics in the treatment of Duchenne and other fatal muscular dystrophies.

Cell Culture:

C2C12 myoblasts and myotubes were grown as previously described. α7^(+/LacZ) myoblasts were originally isolated and maintained as described. Briefly, myoblasts were grown and maintained in DMEM without phenol red (Sigma) containing 20% FBS (Atlanta Biologicals), 1% Penicillin/Streptomycin (P/S) (GIBCO)+L-Glutamine (GIBCO). Myoblasts were maintained below 70% confluence until use in assay. Myoblasts were differentiated into myotubes in DMEM without phenol red, 1% horse-serum (Atalanta Biologicals), and 1% P/S+L-Glutamine. All cells were incubated at 37° C. with 5% CO₂. Assays were performed on myoblasts and myotubes between passages 8 and 14.

Compound Libraries:

Four compound libraries were screened using our muscle cell based assay: 1) Prestwick Chemical and Microsource Spectrum Libraries (BioFocus DPI, Leiden Netherlands with facilities in UK, Basel, Heidelberg) (Overington et al., 2006). 2) DIVERSet library (Chembridge Corp., San Diego, Calif.) and compounds from the ChemDiv library. 3) LOPAC library (Sigma-RPI) consists of 1280 pharmaceutically active compounds. 4) MLSMR-Molecular Libraries Small Molecule Repository.

Myoblast α7^(+/LacZ) Integrin FDG Assay:

A total of 5000 α7βgal^(+/−) myoblasts were dispensed in 100 μL growth media using a 12-well multi-pipette (Rainin) onto Nunc black sided TC coated 96-well plate. After 24 hours up to 1 μl of compound in DMSO was added to the myoblast plates from pre-made working drug plates using a 1 μl 96-well pin tool or using an 8-well automatic multichannel pipette. Each working drug plate contained a column of a positive control (sodium butyrate (Fluka) or SU9516 (Tocris)) and at least one column containing DMSO alone. After incubating for 48 hours the media was aspirated, and cells were lysed with 50 μL of Mammalian Protein Extraction Reagent (MPER) (Thermo) per well followed by incubation at room temperature for 10 minutes. β-galactosidase (βgal) activity in each well was quantified by adding 50 μL of FDG assay solution (20% 0.1M sodium phosphate buffer pH 7.0 (Sigma), 0.2% 1M MgCl₂ (Sigma), 0.2% 20 mM fluorescein di-galactoside (FDG) (Marker Gene Technologies)) and incubating the plates in the dark for 20 minutes at room temperature. Stop solution (2×TE) was then added (100 μl/well) and plates were read for fluorescence on the Victor V (Perkin-Elmer) with an excitation filter at 485 nm, an emission filter at 535 nm, and a 0.1 s/well count time.

Myotube α7 Integrin FDG Assay:

A total of 25,000 α7βgal^(+/−) myoblasts were dispensed in 100 μL growth media. After 24 hours, growth media was aspirated, wells were washed with 200 μL PBS, and 100 μL/well of differentiation media was added. Differentiation media was changed daily between 72 and 120 hours, and up to 1 μL compounds in DMSO were added as previously described once wells contained differentiated myotubes. The FDG fluorescence assay was performed as described in the myoblast screen with the one notable exception being the incubation after FDG solution addition being shortened from 20 minute to 5 minutes at RT due to the higher levels of βgal in myotubes.

qHTS of the LOPAC Library and MLSMR:

A total of 250 α7LacZ^(+/−) myoblasts at passage 13 were dispensed using a mulitdrop (Thermo) into black low base tissue culture treated microclear aurora plates in 6 μl media containing DMEM without phenol, 5% FBS, 1× GluMax and 1× Penicillin/Streptomycin. The plates were incubated 16-24 hours at 37 C, 5% CO2, 95% humidity covered with low evaporation stainless steel lids from Kalypsys. Compounds were then dispensed using a Kalypsys pintool to deliver 23 nl/well compounds in DMSO (diluted into 6 μl resulting in a 1:260 dilution of compound). The positive control compound used was the cdk2 inhibitor SU9516 (Tocris) (identified in the initial LOPAC screen). The plates were incubated for 48 hours at 37 C, 5% CO2, 95% humidity using the same stainless steel lids. After incubation with compound, 5 μl of the media was aspirated using the Kalypsys washer/dispenser and 3 μl of the Mammalian Protein Extraction Reagent (MPER) lysis buffer (Thermo Fisher) was added. The plates were spun at 2000 rpm to remove bubbles and an initial capture was acquired on the Viewlux (Perkin Elmer) with excitation at 480 nm and emission at 540 nm for 25 seconds to omit any auto fluorescent compounds. The plates were then incubated for 10 minutes at room temperature. After incubation, 3 μl of 125 μM FDG (Marker Gene Technologies) diluted in PBS with 2 mM MgCl₂ and 0.2% β-mercaptoethanol (BME) was added, the plates were then centrifuged at 2000 rpms and incubated for 30 minutes at room temperature. The plates were again read on the Viewlux with excitation at 480 nm and emission at 540 nm for 25 seconds.

CMV-LacZ Secondary Screen:

In order to determine compounds that acted to stabilize β-gal in myogenic cells, a CMV-LacZ C2C12 cell line was used in a secondary screen. The EGFP gene from the pEGFP (Clontech) vector was replaced by LACZ PCR product with digests of the vector/PCR product performed using EcoRI (NEB) and NotI (NEB) and ligated using T4 DNA Ligase (NEB) following standard procedures to produce the CMV-LACZ vector. The LACZ gene was generated by PCR from the pBK-RSV (Stratagene) vector using primers LACZ EcoRI F− and LACZ NotI R−. The CMV-LACZ vector was then digested with AfIII (NEB) to linearize the construct, which was then transfected in C2C12 myoblasts using Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Stable cell lines were selected in growth medium containing G418 (Life Technologies) at 1.5 mg/ml. Clonal lines were selected and screened for βgal levels using the myoblast FDG assay. C2C12 cell lines with the highest βgal expression were used to screen cherry picked compounds, following the same procedures defined above for the myoblast and myotube screens. Compounds were determined to have failed this secondary assay if signal was elevated above the average DMSO+2× standard deviation (˜30%) for two separate data points.

Western Blotting:

C2C12 myotubes were treated for 48 hours with each hit compound, washed with PBS, then scraped and pelleted using standard procedures. Cell pellets were resuspended in RIPA buffer and protein concentration determined by BCA. Approximately 10 g of protein was loaded per lane and separated on 8% SDS-PAGE gels and transferred to nitrocellulose using standard conditions. Blots were probed using α7A and α7B integrin specific rabbit polyclonal antibody as previously described and normalized to α-tubulin.

Statistical Analysis and Curve-Fitting:

Statistical analysis was performed using Graphpad Prism software and unpaired t-test comparison against the DMSO control treatment group. Graphpad prism software was also used to fit curves using nonlinear regression analysis with log (agonist) vs. response with a variable slope. A constraint equal to 1 was placed on the bottom of the curve and either 2 or 2.5 at the top (when needed) in order to produce appropriate EC₅₀ values.

Generation and Validation of α7^(+/LacZ) Integrin Myoblast Assay:

In order identify α7 Integrin enhancing compounds, an assay based on α7^(+/LacZ) mouse derived primary myoblasts and myotubes was developed. Heterozygous myoblasts were used in order to maintain a copy of the Itga7 gene for normal myogenic cell adhesion and signaling. The second Itga7 gene allele had exon 1 replaced by the LacZ gene, providing a reporter for α7 integrin transcription and maintaining the endogenous promoter, enhancer, and chromatin environment. By deriving primary muscle cells, myogenic cells capable of differentiating into myotubes and in which the β-gal reporter levels accurately mimicked the α7 integrin protein levels during this developmental process were produced. Several rounds of pre-plating were performed with the myogenic line used for the screen in order to remove fibroblasts, leaving a relatively pure population of myogenic cells.

α7 Integrin Muscle Cell-Based Assay:

Assessing the growth rate of myogenic cells in a 96-well format was carried out. For the myoblast assay, it was determined that plating 5000 α7^(+/LacZ) cells led to a confluence of ˜80% at the time of fluorescence quantification. Limiting the confluence to less than 80% helped prevent entry into a differentiated state in this assay which may affect LacZ expression. For the myotube assay, it was determined that plating 25,000 myoblasts led to a confluence of around 95% after 24 hours allowing myogenic differentiation to proceed. Next, the linearity of fluorescence for the assay was determined for various amounts of purified E. coli β-galactosidase using the FDG assay solution (FIG. 4). This displayed a linear response that was comparable to levels observed in the α7^(+/LacZ) myoblasts. Finally, as library compounds are normally solubilized in DMSO, the effects of DMSO on our assay were examined for both myoblasts and myotubes (FIG. 4). In myoblasts, DMSO concentrations of up to 0.5% had no significant effect on the assay compared to cells without DMSO addition (FIG. 4). At 1% DMSO, there was a 10% decrease in overall fluorescent signal in the assay, which was considered to be within acceptable range (FIG. 4). The effect of DMSO on the myotube assay was more robust, where 0.5% DMSO decreased fluorescence by ˜12% and 1% DMSO displayed ˜25% decrease in overall fluorescence (FIG. 4). Greater than 1% DMSO resulted in reduced fluorescence and therefore 1% DMSO in media was determined to be the maximum acceptable level for myotubes. The levels of FDG were optimized to 12 μM which was used in the MLSMR screen and subsequent verification assays (FIG. 4).

For our preliminary screens, sodium butyrate was determined to increase β-galactosidase levels with treatments of ˜1.5 mM. In initial studies with sodium butyrate a Z′ factor of 0.6, which is considered a strong assay for drug discovery, was calculated and thus a myotube screen of the Prestwick Chemical at 0.2 ug/mL and Microsource Spectrum Libraries at 10 μM was initiated. As these libraries are relatively small, myotubes were initially screened because they are the ideal therapeutic target for α7 Integrin elevation. Using a 1.2 fold increase over DMSO control as the minimum cutoff, 24 compounds in the Prestwick chemical library and 30 compounds in the Microsource spectrum library were identified. These corresponded to a hit percentage of 2.1% and 1.5% of the libraries. After secondary and counter screens, the compounds that increased α7 integrin were classified as either iron chelators or a cholesterol analog. The iron chelators identified in the screen were Ciclopirox ethanolamine, deferoxamine, and 2,2-Dipyridyl which all displayed positive dose-response curves in myotubes. The cholesterol analog compound, 5alpha-cholestan-3β-ol-6-one, also displayed a positive dose-response curve.

This initial success led us to attempt a larger scale high-throughput screen (HTS) using the DIVERSet library of compounds with sodium butyrate as a positive control. Due to the number of compounds in the DIVERSet library (˜50,000), a myoblast screen was performed. This screen generated several more “hits” which upon secondary screens were narrowed down to three compounds annotated: 1001, 1002, and 1003. After further studies, these compounds and analogs were either less robust or too toxic to move forward with further studies.

Primary Screen of LOPAC Library and the MLSMR:

The assay was adapted for a high-throughput screen with the fully automated Kalypsys robotic system using 1536 well plates at NCGC. A preliminary screen of the LOPAC library with 1001 (DIVERSet) as a positive control was used to develop the new conditions for this assay, comparing offline to online results and reducing myoblasts plated to 2000 cells/well for the 384-well format. This screen produced several “hit” compounds including Sodium Nitroprusside dihydrate, a Nitric oxide donor, and two cdk2 inhibitors SU9516 and CK2 inhibitor 2. SU9516 gave a relatively robust response of around 2.1-fold, relative to DMSO alone. There was still a relatively high variability in DMSO background signal (˜30%) even using SU9516 as a positive control. However, as SU9516 was the most reproducibly active drug that had been identified it was selected as a positive control for the qHTS. To this end a dose-response curve SU9516 treatment of myoblasts and myotubes was generated (FIG. 5).

Using SU9516 as a positive plate control and DMSO as a negative control, the assay was then used to screen the Molecular Libraries Small Molecule Repository (MLSMR) of 368,680 compounds at NIH Chemical Genomics Center (NCGC) using concentrations at 0.08 μM, 0.4 μM, 2 μM, and 10 μM. Around 1500 compounds were cherry picked as “hits” based on previously defined curve classifications (FIG. 6).

Offline Confirmation and β-Galactosidase Stability Secondary Screen:

From the 1500 cherry picked “hits” two pools were chosen for further off-line myoblast, myotube and secondary screening. The first pool consisted of 166 compounds, which were still active in a subsequent online myoblast screening of the original 1500 cherry picks (Tables 9-12). The second pool consisted of the top 197 compounds, which had not been repeatedly active in subsequent screens (Tables 9-12). Finally, as SU9516 had proven to be a highly reliable positive control, 44 analogs, based on the SU9516 platform, were selected from the MLSMR library or from separate stock compounds and screened. The secondary screen consisted of a CMV-LACZ stably transfected C2C12 myoblast line with consistent β-Galactosidase activity (Tables 9-16, and Table 17). The assays were used the original 96-well format and new dose-response curves for myoblasts (N=3-6 replicates for each data point), myotubes (N=3-6 replicates for data point), and CMV-LacZ secondary assay (N=2 replicates for each data point) using concentrations of 0.5 μM, 1 μM, 5 μM, 10 μM, 20 μM, and 40 μM (Tables 9-16). A cutoff of greater than 25% relative to DMSO was used to exclude compounds (Table 17). Almost one fifth of the compounds from the initial 197 top “hits” worked exclusively in myotubes assay and not in myoblasts. Together, this data suggested that during the primary screen some of the cells in the assay had differentiated into myotubes. This trend was not observed in the 166 NCGC compounds where almost one third worked exclusively in myoblasts. Again, during the transition from on-line to off-line screening many of the compounds from both groups (54% and 69%, Table 17) failed to achieve the minimum cutoff of 25% increase over DMSO levels in either myoblasts or myotubes. In addition the majority of compounds, which showed activity in the 3-galactosidase stabilizing secondary screen, did not show activity in either of the α7^(+/LacZ) based assays (Table 17, full results in Tables 9-12). These results suggest some of these compounds may not be actually stabilizing or catalyzing the 1-galactosidase enzymatic reaction but instead may be activating the CMV promoter in the C2C12 cells.

As SU9516 was used as our positive control, it was used to potentially represent a platform that could be modified to produce other “hit” compounds. off-line screens with 44 SU9516 analogs were performed. The myoblast, myotube, and secondary CMV-LacZ assays were performed as previously described and results from this screen are summarized in Table 17 (Full results in Tables 13-16). Surprisingly, many of them, as with SU9516 itself, showed activity in the C2C12 CMV-LACZ myoblasts and myotubes. Most of the compounds had activity in the 3-galactosidase stabilizing screen also had no response in the primary screen suggesting that they were not β-galactosidase stabilizers but instead activators of CMV or the region of CMV-LACZ stable integration within this cell line. This result also suggests that SU9516 was likely activating both the ITGA7 promoter and the CMV-promoter by inhibition or activation of separate signaling pathways. With reference to Tables 9-16 below, values of less than about 0.9 correspond to less desirable compounds as compounds with such values were toxic at the indicated concentration value; values of 0.9 to about 1.1 indicate compounds with some activity (substantially similar to the DMSO background); and values greater than about 1.2 indicate compounds that have desirable activity as these values indicate compounds exhibiting an activity about 20% above the background.

TABLE 9 Burkin Assay in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000591667-01 1.1351 0.86235 1.088 1.13163 1.03356 0.99007 MLS000568234-01 1.01115 1.10558 1.37321 1.13643 1.03188 1.09422 MLS000689562-01 0.88167 0.95366 1.22402 1.01263 1.014 1.07412 MLS000732652-01 0.87916 1.0346 1.2648 0.97202 1.19119 1.17557 MLS001240181-01 1.19808 1.15299 1.07353 1.4585 1.08072 1.08233 MLS001211139-01 1.21551 1.218 0.96826 1.06491 1.13586 1.2392 MLS001030268-01 0.60686 0.72598 0.87162 1.48967 1.14329 1.23909 MLS000912699-01 0.43922 0.6724 0.9298 0.93216 1.03847 1.11521 MLS001125260-01 0.90571 1.02411 0.96159 0.98381 1.11006 1.20955 MLS000717689-01 0.98659 0.95999 1.03846 1.03203 1.04195 1.15829 MLS001197665-01 1.18868 1.09447 0.95234 1.01544 1.06912 1.06224 MLS001075922-01 0.64637 0.56776 0.2869 0.60386 1.29157 1.2085 MLS001124046-01 1.07367 1.10106 1.01243 1.07966 1.15704 1.06118 MLS001197220-01 1.0836 1.11129 0.92184 1.84318 1.28622 1.2811 MLS001221318-01 1.07461 1.05387 0.99442 1.00961 1.28345 1.11823 MLS000947910-01 1.01847 1.04651 1.0429 1.03277 1.18767 1.02381 MLS001215795-01 0.71873 0.74097 0.77762 0.93196 1.20673 1.14257 MLS002163670-01 0.7779 0.89724 0.94682 1.01239 1.23366 1.11688 MLS001200149-01 0.98756 1.02136 0.99804 1.0782 1.15573 1.03945 MLS001359861-01 0.91135 0.87253 0.93239 0.94838 0.93763 0.93526 MLS000710669-01 0.82425 1.84768 0.99409 1.0722 1.04969 1.02525 MLS001035690-01 1.09083 1.05474 0.98721 1.18564 1.15072 1.05147 MLS001030621-01 1.00997 1.00129 1.16138 1.15044 1.15833 1.00359 MLS001083082-01 0.94677 1.01412 1.02292 1.05814 0.95159 0.90356 MLS000045588-01 0.89188 1.00313 0.91698 1.07484 1.10703 1.01584 MLS001216939-01 0.78651 0.86335 0.98283 1.11114 1.2827 1.12283 MLS001163859-01 0.92799 0.92082 1.02427 1.05488 1.09001 0.97855 MLS000683232-01 1.65393 1.82061 1.77787 1.6445 1.19868 1.23351 MLS001170856-01 0.71333 0.92168 1.24442 0.97435 0.86511 0.94996 MLS002667707-01 0.83137 0.93474 1.23469 1.01997 0.91716 1.0156 MLS001200665-01 1.02863 1.14332 1.24505 1.00302 0.8539 0.89824 MLS002161853-01 0.9354 1.07259 1.1978 0.93857 0.82784 0.85687 MLS002163101-01 0.97754 0.94568 1.25085 0.96995 0.94422 0.95515 MLS000062431-01 0.9038 1.78859 1.20925 1.01602 0.95817 0.9542 MLS000028160-01 0.85354 1.10681 0.85212 0.98053 0.98728 1.03731 MLS002248819-01 0.76886 0.81835 1.0849 1.06786 1.11265 1.12574 MLS000080654-01 0.91656 1.11208 0.94286 0.92349 0.92297 0.97875 MLS000760876-01 0.95041 1.10218 0.91131 1.81188 0.98558 1.0287 MLS000677675-01 0.95543 1.00416 1.02813 0.98734 1.06053 1.09282 MLS000113985-01 0.12198 0.17793 0.48041 0.78982 1.01847 1.06463 MLS001182368-01 0.89517 1.00108 0.89449 0.91403 1.21452 1.08093 MLS001212882-01 1.04785 1.06823 0.9069 1.04855 1.06975 1.0132 MLS001004364-01 0.93735 0.97287 0.95313 1.03485 1.10452 1.0917 MLS000736846-01 0.95628 1.0406 1.01151 1.06976 1.2858 1.21826 MLS001098105-01 1.13456 1.07472 0.97405 0.99364 1.02871 0.94487 MLS000678673-01 1.13357 1.17252 0.96866 1.04367 1.05388 0.93249 MLS000925023-01 0.41794 0.77654 0.79566 0.95232 1.03462 0.99596 MLS001212319-01 0.78199 0.85293 0.88263 0.94115 1.02595 1.04963 MLS000779126-01 0.93895 0.91244 0.81956 0.92292 1.11482 1.01879 MLS000948055-01 0.58675 0.82128 0.8986 1.13571 1.05347 1.32788 MLS000110418-01 0.8889 1.00365 1.11988 1.24866 1.07925 1.3095 MLS000693704-01 0.80188 0.84732 0.94181 1.1427 1.15286 1.42821 MLS001225512-01 1.28384 1.10796 0.88323 1.17276 0.91313 1.1596 MLS001006798-01 1.01583 1.11843 1.00833 1.13293 0.92818 1.19095 MLS000711491-01 0.57934 0.79492 1.02377 1.02034 1.0159 1.21405 MLS000582947-01 0.77374 0.9417 0.98066 0.94304 0.96323 1.23612 MLS000531177-01 0.80079 1.01406 1.62515 1.06657 0.92304 0.88259 MLS001202389-01 0.25563 0.58161 1.1004 1.00911 0.94807 0.9323 MLS000536064-01 0.45011 0.54402 1.04652 0.73332 0.80395 0.80279 MLS000586245-01 1.02068 1.03337 1.24403 1.11633 1.04937 0.99033 MLS001061374-01 0.67743 0.87297 1.16907 0.98264 0.84475 0.84766 MLS000675441-01 0.89965 0.90155 1.24817 1.04359 0.8764 0.93971 MLS001200396-01 0.4794 0.48269 1.1321 0.53784 0.92449 0.88271 MLS001165937-01 0.68201 1.03268 1.70065 1.27472 0.89489 0.86889 MLS000325736-01 0.88542 0.91197 1.16957 1.42277 0.78755 0.77572 MLS001215357-01 0.53275 0.9203 1.48859 0.88374 0.93 1.03703 MLS000588210-01 0.96107 1.13586 0.98164 0.90019 0.97069 1.05483 MLS000764729-01 0.72603 0.9581 0.96935 0.98159 1.0582 1.08805 MLS000689492-01 0.96521 1.08264 0.94309 0.93998 0.90795 1.07297 MLS001000299-01 0.77383 0.93971 0.97497 0.95516 0.99529 1.08932 MLS000393762-01 0.46958 0.59613 0.69566 0.77991 0.94283 0.97379 MLS001130011-01 0.91554 1.03734 0.97619 1.37412 0.89472 0.97145 MLS001229477-01 0.90194 0.96342 0.94542 1.36703 0.82957 0.87031 MLS000707378-01 1.49252 1.31687 1.07579 1.02429 1.04559 1.00348 MLS000573208-01 0.62351 1.63233 0.81207 0.95231 1.01493 1.03104 MLS001167281-01 0.86182 0.88689 0.97761 1.07478 1.18016 1.08931 MLS000053342-01 1.02205 1.0354 0.86681 0.97655 1.08832 1.22195 MLS002171615-01 0.61782 0.70899 0.88587 0.97469 0.94068 1.05229 MLS001005712-01 0.76395 0.87627 0.87827 0.92598 1.17003 1.1047 MLS001176153-01 1.12011 1.14544 0.87665 0.91849 1.04463 1.06004 MLS000735021-01 0.99412 1.02509 0.97651 0.99818 0.92462 0.94018 MLS000767397-01 0.97544 1.61335 0.93339 1.47579 0.83903 0.88624 MLS001196572-01 0.9345 0.93622 0.94826 1.22354 0.76263 0.88263 MLS000393966-01 0.96498 0.99041 1.07573 1.79279 0.96263 0.94947 MLS001034810-01 0.84068 0.8996 1.19071 1.37466 0.99747 1.02099 MLS001165394-01 1.00709 1.03548 0.95105 1.15909 1.20071 1.14858 MLS000089464-01 0.39111 0.56309 0.7825 0.93396 0.98019 0.99651 MLS000698617-01 0.89657 0.84622 0.93172 1.06001 1.00295 1.00774 MLS001175021-01 0.95749 0.97466 0.90603 0.88063 0.98034 0.92001 MLS001166758-01 0.61737 0.72753 0.82445 0.97363 0.73944 0.82402 MLS001008109-01 0.83059 0.82603 0.86263 1.00193 0.78003 0.84815 MLS001181936-01 0.32845 0.50999 0.82189 0.85734 0.75618 0.84427 MLS000560266-01 0.55892 0.83206 1.13014 1.02573 0.85564 0.95298 MLS001215074-01 0.59739 0.73389 0.97384 0.94307 0.9004 1.02349 MLS001215123-01 0.52524 0.665 1.15451 1.1241 0.97081 1.07545 MLS001033255-01 0.82795 0.98503 1.12416 1.01684 0.93725 1.01302 MLS001160611-01 0.92674 0.97495 1.11549 1.00379 0.97673 1.1058 MLS001006302-01 0.94141 0.97305 1.17248 0.95352 0.93033 1.12331 MLS001123876-01 0.88436 0.93393 1.09536 1.43977 0.82637 1.04105 MLS001122698-01 0.83886 0.9061 0.96519 1.00842 0.85728 0.98289 MLS000755214-01 0.86097 0.84475 0.96348 0.97565 1.02077 1.07667 MLS000731285-01 0.60769 0.60402 0.7344 0.77267 0.92346 1.06384 MLS000776409-01 0.34443 0.50867 0.98382 0.93888 1.00738 1.1194 MLS001221908-01 0.56836 1.43328 0.88118 0.93061 0.95314 1.05089 MLS000419286-01 0.71995 0.7945 0.8951 0.91739 1.00244 1.16649 MLS000554416-01 0.82485 1.08704 0.9591 0.99152 1.0679 1.21133 MLS000073150-01 1.06802 1.13888 0.90898 0.92622 1.0875 1.26179 MLS000663185-01 0.83278 0.92216 0.93123 1.39854 0.97437 1.10863 MLS001078811-01 0.74109 1.68071 0.89103 0.95834 0.99261 0.9376 MLS002694363-01 0.70661 0.75811 1.08986 1.04452 1.06141 1.06241 MLS000689218-01 1.03748 1.07006 1.05082 1.13165 1.17068 1.06835 MLS001215294-01 0.20458 0.20253 0.61411 0.76405 1.00264 0.97828 MLS001183575-01 0.53384 0.69163 0.79465 0.89805 1.00931 0.96443 MLS000393567-01 0.87054 0.83339 0.93251 0.97236 1.05083 1.03233 MLS000546316-01 1.02675 1.02594 1.12291 1.16412 1.22254 1.15942 MLS000912258-01 0.37249 0.63513 0.84251 1.2722 0.90885 0.84356 MLS000850522-01 0.88047 0.8743 1.00276 1.30679 0.93316 0.79932 MLS001197779-01 0.75302 0.84665 0.98695 1.28053 1.0032 0.91613 MLS001095705-01 0.72839 0.79174 0.969 1.29549 0.87876 0.78726 MLS000912562-01 0.55515 0.69112 0.92532 1.03872 0.92904 0.76968 MLS000092785-01 0.6509 0.78329 0.93433 1.1703 1.03537 0.95442 MLS000418650-01 0.94454 0.91042 0.96326 1.12987 1.06885 0.91564 MLS000860966-01 1.11657 1.24227 1.12552 1.22473 0.95061 0.8092 MLS001180929-01 1.1941 1.10157 1.05028 1.04982 1.0555 1.07667 MLS000564564-01 0.97011 0.94044 1.0834 1.04244 0.94502 0.99186 MLS001164432-01 0.66217 0.7137 1.06663 1.09125 1.1813 1.24825 MLS000621451-01 1.08838 1.0713 1.11408 1.06613 1.01829 1.06548 MLS000078709-01 0.26162 0.51714 0.80516 0.81574 0.996 1.06264 MLS000688267-01 1.10743 1.0466 0.91328 1.35814 1.10445 1.09233 MLS001006516-01 0.48019 0.69276 0.79316 0.8796 1.13422 1.09738 MLS001095033-01 1.0618 1.12466 0.96135 0.96322 1.18613 1.28147 MLS000072290-01 0.22377 0.39842 0.6517 0.73337 1.19579 1.40542 MLS000036988-01 0.65813 0.90234 0.82559 0.86568 1.0825 1.18898 MLS001217935-01 0.58698 0.80022 0.89296 0.91061 1.07572 1.16224 MLS000777780-01 0.72314 0.91334 1.24779 0.94395 1.05647 1.13609 MLS001174740-01 0.54139 0.96576 1.18066 0.85119 1.19298 1.22104 MLS000392555-01 1.22568 1.31366 0.97342 1.30012 1.10052 1.2055 MLS000693795-01 1.24042 1.18741 0.97277 1.02032 1.24179 1.1137 MLS000684034-01 0.30855 0.42247 0.62584 0.78721 1.01964 0.99899 MLS001172822-01 0.9597 0.98696 0.90137 0.96623 1.35133 1.23301 MLS000052969-01 0.75911 0.82915 0.83559 0.96455 1.35174 1.24959 MLS001217212-01 1.06402 0.99668 0.99332 0.97848 1.17844 1.09519 MLS001004864-01 0.88826 1.10454 1.02341 1.10852 1.19287 1.1396 MLS001116535-01 1.26741 1.23894 0.93856 0.99796 1.11141 1.09197 MLS001165424-01 1.22991 1.17612 0.95477 0.99659 1.2222 1.15184 MLS001116079-01 1.34036 1.24425 0.9749 1.02479 1.08671 1.02952 MLS001198271-01 1.0038 1.08798 1.03423 1.29177 1.00045 1.03412 MLS001167798-01 1.00561 1.05069 1.06388 1.27705 1.16574 1.05836 MLS000710288-01 0.59627 0.86808 1.1778 1.3928 1.30459 1.16807 MLS000734270-01 0.98726 0.97392 0.95501 1.21022 1.04793 1.0135 MLS000858981-01 1.00476 1.0113 1.01009 1.18572 0.999 0.95297 MLS000698826-01 1.02313 0.99365 0.95981 1.24627 1.00831 1.01911 MLS001000874-01 0.95449 1.00725 0.86772 1.07723 1.03283 1.17509 MLS000682750-01 1.10263 1.12414 1.07554 1.22927 1.02005 1.20347 MLS001090787-01 1.22203 1.26522 1.45989 1.26261 0.97343 1.14195 MLS002636056-01 0.82025 0.93768 1.22389 1.03743 1.06589 1.15719 MLS002170630-01 0.67353 0.78253 1.21616 1.0982 0.97082 1.12201 MLS002162890-01 0.49245 0.694 1.26109 1.1338 1.03367 1.10651 MLS001105912-01 0.46309 0.60252 1.24569 1.02884 1.05749 1.16179 MLS001007892-01 0.49826 0.72245 1.22958 1.01568 1.03672 1.12717 MLS000089748-01 0.17663 0.24025 0.73549 0.86559 1.16763 1.14515 MLS000912726-01 1.02179 1.1258 1.02001 0.98384 1.17723 1.27931 MLS000086970-01 0.78111 0.8541 0.87225 0.85834 1.07424 0.98177 MLS000420298-01 0.92353 1.11099 1.10015 1.08324 1.24343 1.37886 MLS001147478-01 0.82145 0.9651 1.19218 1.0569 1.09145 1.14192 MLS000090135-01 0.39561 0.60292 1.01787 0.98804 1.16609 1.22735 MLS001179717-01 0.05093 0.30517 0.67937 0.83575 0.86405 0.91197 MLS000683234-01 1.21046 1.06733 1.15246 1.13392 0.83414 1.18747 MLS000695955-01 1.03417 0.96102 1.06308 1.08042 0.87081 0.90171 MLS001125488-01 0.92319 0.9073 0.87883 0.9899 0.97552 0.93329 MLS000768008-01 0.87872 0.78367 0.87683 0.89762 0.96178 0.9886 MLS000913117-01 0.05713 0.19517 0.67292 0.91381 0.91552 0.90847 MLS000860538-01 0.97828 0.958 1.11473 1.07765 1.03288 1.02022 MLS001177259-01 0.77735 0.76937 0.91654 0.97354 0.98957 0.96057 MLS000861434-01 0.93215 0.87769 0.97717 0.91327 1.02784 0.94636 MLS000047918-01 0.78033 0.80348 1.0006 0.94829 0.91191 0.87328 MLS000389484-01 0.61319 0.73315 0.8706 0.85238 0.76721 0.78483 MLS001217673-01 0.80793 0.83254 0.92563 0.93529 0.83431 0.84936 MLS000389677-01 0.60885 0.66194 0.79763 0.84263 0.88312 0.53248 MLS001208858-01 0.84248 0.85481 0.87594 0.88923 1.00324 0.9851 MLS000333610-01 0.77941 0.79748 0.91885 1.04819 0.92531 0.8664 MLS001117351-01 0.4323 0.71365 0.74454 0.77183 1.10521 1.10312 MLS000682883-01 0.73791 0.82691 0.91923 1.01077 0.96331 0.94759 MLS001095231-01 0.72802 0.91969 0.86365 0.92419 0.96824 0.96919 MLS000721584-01 0.7628 0.86626 0.98462 1.02979 0.85252 0.933 MLS001183429-01 0.33287 0.56663 0.73733 0.78206 0.66516 0.75919 MLS002158881-01 1.21172 1.11559 1.01779 0.98858 0.83776 0.88812 MLS001165749-01 0.77107 0.96276 0.93419 1.03408 1.05712 1.04807 MLS001237320-01 0.81867 0.92872 0.84952 0.91452 0.98478 0.97334 MLS000763405-01 1.083 1.469 1.53163 1.46329 1.03014 0.96126 MLS000538580-01 0.02896 0.03863 0.53063 0.8691 0.90971 0.96452 MLS001033202-01 0.77044 0.84083 0.92815 0.99521 0.91528 0.66817 MLS001216260-01 0.89335 0.87813 0.87981 0.8531 0.98403 1.0145 MLS000085522-01 0.74815 0.97285 0.95306 1.11804 0.87205 0.99272 MLS000702680-01 0.62643 0.69252 0.89075 0.86233 0.8804 0.84503 MLS001212998-01 1.29981 1.36467 1.54392 1.45225 1.19984 1.19252 MLS001160885-01 0.79137 1.02135 0.91473 0.94021 0.93557 1.05034 MLS001122718-01 0.93319 0.99226 0.90919 1.03265 1.04066 1.1056 MLS000027478-01 0.81604 0.91452 0.92087 0.97213 0.90438 0.95522 MLS001177364-01 0.83857 1.00465 0.90148 0.96554 0.98693 1.08229 MLS001179695-01 0.80683 0.89235 0.98712 0.87525 0.94414 1.03309 MLS002251986-01 0.91837 0.97926 0.86667 0.95123 0.92314 1.04571 MLS001166704-01 0.43935 0.57467 0.76522 0.84987 1.16515 1.10905 MLS001196422-01 1.00409 1.24784 1.33031 1.22893 0.9784 0.74578 MLS001179624-01 0.88278 0.87902 0.93143 0.95146 0.99629 0.99296 MLS001223425-01 1.1318 1.22256 1.14626 1.05531 1.08325 1.05664 MLS001117140-01 0.94202 0.9222 0.90096 0.90913 1.06544 1.02935 MLS001110618-01 1.08304 0.97133 1.00452 0.9228 1.02989 0.95251 MLS001223482-01 0.98024 1.28967 1.02296 1.0127 1.09469 1.06378 MLS000680049-01 0.63536 0.74931 0.9727 0.96933 1.10296 0.99975 MLS001212498-01 1.39711 1.28613 1.31206 1.18111 1.20238 1.05365 MLS001124732-01 0.65809 0.68925 0.81246 0.8622 1.07116 1.12082 MLS000526364-01 1.02864 1.11416 1.08517 1.04544 0.95015 0.97473 MLS000767227-01 0.92827 0.91325 0.91067 0.85772 0.80731 1.02529 MLS000703499-01 0.10433 0.44649 0.76322 0.9278 1.01689 0.70927 MLS001167169-01 0.90615 0.81331 0.93783 0.95467 0.8858 1.03215 MLS001198693-01 0.91638 0.93507 0.91367 0.92293 0.54265 0.96167 MLS001219345-01 0.92275 0.98071 1.01406 1.04565 1.11947 0.30269 MLS001211651-01 1.02624 0.99093 0.97202 1.01085 1.12405 1.06095 MLS000806880-01 1.05558 0.99571 0.95237 1.00906 1.12862 1.09682 MLS001223567-01 0.86396 0.85795 0.92033 0.96076 1.08244 1.04444 MLS001005283-01 0.93009 0.89649 0.89081 0.93463 0.85822 0.87024 MLS001218427-01 0.30387 0.22324 0.76631 0.83344 0.94853 0.9466 MLS001139288-01 0.93365 1.08484 0.92246 0.86341 0.97087 0.98227 MLS000696445-01 0.80698 0.81296 0.88069 0.86702 0.56209 1.01258 MLS001218795-01 0.98756 0.93531 0.91165 0.86087 0.95964 1.02373 MLS000419555-01 0.79662 0.94775 1.02881 1.06624 1.20411 1.18538 MLS001225507-01 0.88802 0.86847 0.8292 0.90357 0.94438 1.0374 MLS000663651-01 1.04765 0.95036 0.95509 0.98337 1.05878 1.14296 MLS000706349-01 0.65331 0.85838 0.84341 0.85931 0.97973 1.08607 MLS000393110-01 0.71005 1.01204 1.08776 1.07374 0.92729 0.90455 MLS000574647-01 0.83429 0.89851 0.95152 0.96407 1.02749 1.08127 MLS000532969-01 1.77036 1.94964 1.66961 1.43381 1.25091 1.2092 MLS001125260-01 0.84255 0.87384 0.88933 0.9248 1.02145 1.07071 MLS000122749-01 0.94804 1.18542 1.11587 1.02606 1.03955 1.11255 MLS001150751-01 0.84542 1.07873 0.95726 0.99415 1.059 1.11416 MLS001221867-01 0.89431 0.96155 0.92196 0.92746 1.02402 1.00113 MLS001147727-01 0.90442 1.12838 0.88917 1.02852 1.03907 1.21211 MLS000688437-01 0.80643 0.8203 0.90766 0.82332 0.88782 0.89005 MLS001211976-01 0.89518 0.94171 0.91545 0.90182 0.94602 0.95829 MLS002161350-01 0.82621 0.822 0.95139 0.95314 0.92361 0.94036 MLS001077207-01 1.14734 1.01291 1.06263 1.05309 1.0107 0.96989 MLS001209245-01 1.03855 1.09982 1.07082 1.02132 1.13168 1.03871 MLS000737953-01 1.23263 1.14945 1.07964 1.00275 0.96727 0.89998 MLS000552080-01 0.88548 0.93327 1.089 1.05564 0.97843 0.96895 MLS000737204-01 0.74718 0.87311 0.93361 0.91643 0.98155 0.90109 MLS000579238-01 0.9117 1.10725 1.30359 1.16711 1.11338 0.96109 MLS001181671-01 0.89104 0.81134 0.86384 0.87588 0.96504 1.05191 MLS001167424-01 0.81643 0.7995 0.90704 0.89245 1.42763 0.99036 MLS000094770-01 0.6762 0.79989 0.98661 0.94914 0.96764 1.02063 MLS001123810-01 0.87288 0.8853 0.97786 0.97821 0.95241 0.97668 MLS000532078-01 1.07709 1.09043 0.94314 0.94396 1.07555 1.09068 MLS000585616-01 0.93184 0.93498 0.97673 1.00788 0.94346 0.68079 MLS000553673-01 0.88921 0.9043 1.0007 1.03788 0.89516 1.00886 MLS001175592-01 0.94054 0.97307 0.90064 0.93232 0.88593 0.89891 MLS001033255-01 0.79904 0.79618 0.95423 0.95091 0.9407 0.94675 MLS000733703-01 0.8463 0.86346 0.87341 0.89191 0.95923 0.98802 MLS001096269-01 1.19809 1.0523 1.02918 0.94134 1.01068 1.04248 MLS001162872-01 0.7773 0.78298 0.904 0.90246 0.94902 0.95468 MLS000584511-01 1.07248 1.04995 1.08732 1.03293 0.88848 0.5425 MLS001166325-01 1.03177 0.97718 0.94529 0.9487 0.9268 1.01725 MLS000122180-01 0.91163 1.00352 0.96652 0.97257 0.84241 0.80232 MLS001157804-01 1.10384 1.05163 1.11684 0.99798 0.93852 0.87895 MLS000693729-01 0.91945 0.91361 0.90877 0.86473 0.7902 0.90691 MLS001220669-01 0.76127 0.87013 0.9605 0.90157 0.94259 1.00023 MLS001214461-01 0.76704 0.84718 0.81234 0.89639 0.92407 1.06371 MLS000081838-01 0.92338 0.98223 0.92525 0.96114 1.04698 1.0627 MLS002402866-01 0.76886 0.8943 0.93757 0.9113 1.1052 1.03727 MLS001200980-01 0.92013 0.95613 0.95438 1.05452 0.92555 0.99175 MLS001174719-01 0.52655 0.75243 0.84804 1.01314 1.04779 1.05338 MLS001175449-01 0.75639 1.06596 0.99486 1.04541 1.03595 1.0562 MLS001172577-01 0.94305 1.0212 1.0758 1.14243 0.96541 0.99596 MLS001122792-01 0.80584 0.99893 0.8643 0.91298 0.87873 0.97016 MLS001216405-01 0.76668 0.92023 0.92084 0.9945 0.97845 1.13166 MLS000673766-01 0.99188 0.91 1.0253 0.92796 1.06126 1.02043 MLS000912614-01 0.77984 0.73821 0.93996 0.95339 1.08794 0.95511 MLS001194551-01 1.39229 1.17648 1.29698 1.11537 1.20463 1.09593 MLS001214704-01 1.04984 0.9848 1.18552 1.07694 1.29585 1.11409 MLS001219159-01 0.95198 0.91989 0.96435 0.91609 1.059 0.95972 MLS001060549-01 0.94107 0.77307 1.07213 1.01176 1.20369 1.05617 MLS000850824-01 1.06134 0.98611 1.07604 1.07622 1.27474 1.17361 MLS000879190-01 1.12798 1.09398 1.37657 1.18531 1.34461 1.09674 MLS001163140-01 0.98293 0.93502 1.09843 0.99656 1.21719 1.01896 MLS000046123-01 0.98939 1.00216 1.00659 0.95652 0.98085 1.049 MLS000086970-01 0.93423 1.01907 0.95467 1.60052 1.19781 1.01404 MLS000388722-01 0.94068 0.91005 1.1175 1.1052 1.1187 0.93002 MLS000676974-01 0.51838 0.66732 1.09948 0.98998 1.13674 1.10496 MLS000772580-01 0.72509 0.79384 0.8513 0.85541 0.97361 0.84867 MLS000698686-01 0.82518 0.84559 1.07491 1.05367 1.01783 1.03691 MLS001162337-01 0.81495 0.87886 1.07141 1.09144 1.12717 1.12285 MLS001204005-01 1.1735 1.23109 1.23509 1.02172 1.15194 1.0842 MLS000721525-01 0.98502 0.99363 1.00993 0.98343 1.08578 1.08628 MLS000525404-01 1.79482 1.4553 1.28085 1.12043 1.08413 0.99959 MLS000772194-01 1.14873 1.09796 1.08185 1.05483 1.05904 1.09583 MLS000775793-01 0.98952 0.9292 1.07565 1.10917 1.07613 1.10346 MLS000710130-01 0.86888 0.83263 1.03606 1.00611 1.10306 0.96656 MLS001146463-01 0.80609 0.73177 0.92926 0.90269 0.97806 1.02615 MLS000712769-01 0.78927 0.76968 0.92863 0.94757 1.05857 1.05487 MLS000334464-01 1.07573 0.8957 1.14233 1.17793 1.11026 1.12515 MLS000862690-01 0.97527 0.87171 1.00387 1.00872 1.02652 1.02904 MLS001179267-01 0.94996 0.93048 0.9902 0.97969 1.03143 0.98302 MLS000683174-01 0.80586 1.02775 1.01393 1.01994 0.91756 1.01961 MLS000913052-01 1.20186 1.11329 1.06715 1.07591 1.03833 1.11596 MLS001080869-01 0.84846 0.97296 0.9857 1.14722 1.03572 1.14407 MLS000332693-01 0.92254 0.89498 1.09531 1.06767 0.99365 1.03957 MLS001141113-01 0.55984 0.91514 1.07109 1.07883 1.058 1.22948 MLS001176611-01 0.91345 0.83982 1.07215 1.09667 0.99398 1.04935 MLS001202627-01 0.21788 0.54268 0.77391 0.99233 1.08133 1.17187 MLS000765108-01 1.02593 1.04146 1.11307 1.20183 1.1197 1.17146 MLS000937079-01 0.99561 1.02838 1.14674 1.05254 0.89583 1.16289 MLS001215742-01 0.81646 0.76822 1.10465 1.04044 0.92602 0.88704 MLS001217045-01 1.10891 1.05719 1.11177 1.08675 0.96972 0.88104 MLS001196946-01 0.08385 0.11943 0.61881 0.79887 0.97976 0.95406 MLS001216714-01 0.27369 1.08646 1.13942 1.04321 0.97056 1.018 MLS000772430-01 0.89631 0.88124 1.04602 1.00717 0.93622 0.97283 MLS000693370-01 0.93667 0.95143 1.06427 0.88727 0.80004 0.86381 MLS000769322-01 0.95504 1.00606 1.0771 0.96529 0.93537 0.95348 MLS000721030-01 1.12244 1.02526 1.16581 1.02742 0.93485 0.9673 MLS001176897-01 0.94746 0.94581 0.99783 0.90579 0.94565 0.9342 MLS000774940-01 0.77609 0.85802 1.04275 1.09508 0.89283 0.93089 MLS001030746-01 0.96367 0.93385 1.07333 1.06913 0.8373 0.91954 MLS003126425-01 0.44013 1.12959 1.254 1.28169 1.1398 1.14604 MLS001217697-01 1.0127 0.88306 1.15767 1.18252 1.11591 1.09067 MLS000516719-01 0.96515 0.91967 1.06775 1.0321 1.04756 1.00574 MLS001165323-01 0.47791 0.87431 0.92404 0.88215 0.94909 0.97044 MLS001220803-01 0.99059 0.95026 0.95106 0.88777 1.0343 0.96878 MLS001163121-01 0.91572 0.80739 0.99067 0.94885 1.01191 1.04852 MLS001060561-01 0.63183 0.79502 0.88777 0.82662 0.92837 0.99611 MLS001139515-01 0.85269 0.83359 1.15988 1.05338 0.98522 0.92246 MLS001149811-01 0.61724 1.00026 1.02244 1.01027 0.88883 0.64967 MLS000773700-01 0.87206 0.9227 0.86573 0.90585 1.0257 0.93354 MLS001177045-01 0.95693 1.03087 1.0259 1.0025 1.03211 0.84633 MLS000693747-01 0.91746 0.87957 1.08763 1.0596 1.03615 1.11428 MLS001175556-01 0.80176 0.89561 0.88558 0.92019 0.86252 0.92004 MLS001175473-01 0.86983 0.899 0.87154 0.87321 0.37213 0.53313 MLS002156278-01 0.82456 0.86044 0.96531 1.00963 0.96419 0.85974 MLS000707281-01 0.79693 0.83587 0.88542 0.89346 0.97563 0.95067 MLS000591198-01 0.47304 0.77654 0.95931 1.03702 0.92511 1.00578 MLS000714175-01 0.96263 0.99361 1.06 1.1625 0.97498 0.53831 MLS002163386-01 0.93876 1.01374 0.88796 1.03415 0.9023 1.0508 MLS000761297-01 0.95256 1.07174 1.05527 1.12127 1.07133 1.0693 MLS002245351-01 0.86569 0.90007 0.97044 1.03385 1.03691 1.08244 MLS000718886-01 0.81389 0.91243 0.96613 1.08727 0.93943 1.05198 MLS002156485-01 1.02644 1.05206 0.87521 0.92779 0.9382 1.05917 MLS001140657-01 0.79783 0.85676 0.87677 0.95283 0.96198 0.9918 MLS002157024-01 1.08144 1.09694 0.85235 0.96094 0.97128 1.01144 MLS000721730-01 0.93424 0.82698 0.88242 0.81574 0.81973 0.83765 MLS000705922-01 0.87835 0.88922 0.72832 0.72819 1.02 0.96273 MLS000724709-01 0.55365 0.48598 0.77902 0.73891 0.95538 0.98992 MLS002161757-01 1.15126 1.00861 0.91351 0.91126 0.89196 0.96138 MLS002164687-01 0.81941 0.83778 0.84902 0.69489 0.81935 0.77498 MLS001060533-01 1.21969 1.39744 1.10126 1.03286 0.99152 0.90492 MLS000685139-01 0.98483 0.98933 0.77595 0.84163 0.6285 0.82342 MLS001217286-01 1.06759 1.00563 0.82726 0.76705 0.8506 0.74275 MLS001221619-01 0.89756 0.96481 0.67637 0.73674 0.76333 0.68552 MLS001219621-01 0.54233 0.78837 0.75418 0.72486 0.80633 0.86421 MLS001166156-01 0.34807 0.75715 0.76012 0.75432 0.90852 0.8005 MLS000534926-01 0.42215 0.59957 0.88325 0.85863 0.94733 1.52217 MLS000548725-01 0.07694 0.51687 0.76446 0.89891 0.88938 0.91317 MLS000374261-01 0.83211 0.83763 0.71381 0.72479 0.77583 0.7676 MLS000123454-01 1.01341 1.51865 0.86209 0.78618 0.8234 0.8934 MLS000625140-01 0.68287 0.75893 0.78335 0.87481 0.8709 0.88394 MLS001214443-01 0.9313 0.9132 0.81732 0.7787 0.80738 0.92033

TABLE 10 Burkin Assay in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000591667-01 0.69646 0.67806 0.99863 1.07156 1.1434 1.00531 MLS000568234-01 0.97051 0.86942 1.08971 0.96419 1.16742 1.07977 MLS000689562-01 0.85465 0.72357 1.0159 0.97883 1.03466 1.08068 MLS000732652-01 0.73092 0.67974 1.14125 1.1938 1.11599 1.09214 MLS001240181-01 0.79047 0.88714 0.96827 1.64271 1.214 1.17431 MLS001211139-01 0.75468 0.82834 0.94255 1.00287 1.28848 1.16726 MLS001030268-01 0.76063 0.62773 0.92494 1.21276 1.16147 1.07576 MLS000912699-01 0.65181 0.52169 0.93901 0.92519 0.93756 1.17498 MLS001125260-01 0.74508 0.76341 1.09105 0.96768 1.20801 1.28483 MLS000717689-01 0.71346 0.82829 1.07589 1.04848 0.90065 1.11085 MLS001197665-01 0.95828 1.00065 0.99899 0.97302 1.06784 1.11547 MLS001075922-01 0.82425 1.06745 0.62896 0.65563 1.14344 1.29247 MLS001124046-01 0.91979 0.89276 1.01603 1.41708 1.09565 1.14517 MLS001197220-01 0.83097 0.90004 1.00438 1.25409 1.3061 1.32659 MLS001221318-01 0.7186 0.80714 0.95914 0.92662 1.08244 1.13276 MLS000947910-01 0.95151 0.96285 1.03459 0.99998 1.10147 1.13447 MLS001215795-01 0.88728 0.9147 0.91066 0.86305 1.19109 1.29108 MLS002163670-01 0.73497 0.85264 1.07165 0.97782 1.29363 1.22297 MLS001200149-01 0.83633 0.92859 1.09373 1.06886 1.07233 1.26026 MLS001359861-01 0.80395 1.0337 0.876 0.90881 1.18375 1.09515 MLS000710669-01 1.0073 0.99667 0.90001 1.05381 1.16308 1.04515 MLS001035690-01 0.92376 0.97197 0.93577 1.07274 1.07159 1.11088 MLS001030621-01 0.72029 0.7954 0.94292 0.99904 0.94925 0.97239 MLS001083082-01 0.93703 0.89872 1.05034 1.07008 0.94106 1.03453 MLS000045588-01 0.95463 1.06301 1.03566 1.135 1.09854 1.07688 MLS001216939-01 0.47431 0.85294 1.06002 1.06991 1.18531 1.19276 MLS001163859-01 1.05788 1.11822 1.24934 1.38468 1.28077 1.13173 MLS000683232-01 1.31969 1.4149 1.44782 1.27158 1.2778 1.13572 MLS001170856-01 1.02912 0.77626 1.06447 0.91001 0.74357 0.82959 MLS002667707-01 0.7617 0.80494 0.95401 0.89392 1.01022 0.99551 MLS001200665-01 0.88772 0.83653 1.12552 1.09065 0.84612 1.03137 MLS002161853-01 0.93534 0.91896 1.15981 0.98887 0.80923 0.87612 MLS002163101-01 0.97609 0.77823 1.30157 1.10149 0.90746 1.04549 MLS000062431-01 0.88412 0.76433 1.1989 1.17804 0.84151 0.95709 MLS000028160-01 0.81118 0.90493 0.95982 0.91617 1.10188 1.08464 MLS002248819-01 0.85083 0.92428 1.24039 1.06554 1.19767 1.11701 MLS000080654-01 0.62169 0.74219 0.97069 0.90438 1.09159 1.06711 MLS000760876-01 0.89931 0.86336 0.9755 1.24882 1.25578 1.2427 MLS000677675-01 0.68734 0.66754 1.04369 1.08103 1.20496 1.27844 MLS000113985-01 0.66792 0.76552 0.99254 0.99189 1.03688 0.98825 MLS001182368-01 0.91934 0.90974 1.02027 1.2117 1.0792 1.04351 MLS001212882-01 0.91073 0.84206 0.8877 0.90721 1.02311 0.98143 MLS001004364-01 1.05525 0.88751 0.86169 0.90474 1.10504 1.09276 MLS000736846-01 0.96729 0.87764 0.93964 1.00375 1.14446 1.13093 MLS001098105-01 0.84588 0.81422 0.83539 0.91598 1.05323 1.05204 MLS000678673-01 0.78099 0.86554 0.96062 0.97673 0.98018 0.97725 MLS000925023-01 0.73184 0.80043 0.8784 0.89694 1.23995 1.16045 MLS001212319-01 0.63105 0.73964 0.92007 0.94764 1.13867 1.13422 MLS000779126-01 0.77678 0.88319 0.87581 0.91457 1.01779 0.98357 MLS000948055-01 0.75145 0.98002 0.8264 0.90529 1.05415 1.06716 MLS000110418-01 0.88548 0.99394 0.943 1.01658 1.0975 1.07264 MLS000693704-01 0.82627 0.9587 0.92299 1.00259 1.0787 0.93149 MLS001225512-01 0.74015 0.84309 0.82346 0.97987 1.05854 0.92901 MLS001006798-01 0.88856 0.97456 0.89057 1.05957 1.04218 0.93455 MLS000711491-01 0.57898 0.67103 0.94615 0.9629 1.06241 1.06977 MLS000582947-01 0.79101 1.10221 0.97595 1.01969 1.15528 0.90028 MLS000531177-01 0.90051 0.80069 1.16345 1.05071 0.73291 0.82589 MLS001202389-01 0.55936 0.64125 1.05346 0.99362 0.93925 0.9181 MLS000536064-01 0.7073 0.76421 1.13143 0.95394 0.95554 0.90458 MLS000586245-01 1.02629 0.94257 1.17916 1.04311 0.9584 1.00329 MLS001061374-01 0.81546 0.73205 1.18149 1.00437 1.09204 1.05776 MLS000675441-01 0.71678 0.74757 1.11902 0.95908 1.07053 0.85516 MLS001200396-01 0.71138 0.6408 1.26379 0.98293 0.94875 0.93009 MLS001165937-01 0.72485 1.00559 1.52391 1.21196 1.16352 0.96798 MLS000325736-01 0.69718 0.6582 1.16824 1.02677 0.80167 0.91355 MLS001215357-01 0.69286 0.75605 0.88397 0.90573 0.81367 0.9366 MLS000588210-01 0.95653 0.97629 0.91553 0.94662 0.95586 0.94246 MLS000764729-01 0.60751 0.68098 0.97198 1.00794 1.08392 1.05496 MLS000689492-01 0.75436 0.88357 0.92917 0.93108 1.13846 1.15571 MLS001000299-01 0.55782 0.793 0.91625 0.87644 1.00592 0.92249 MLS000393762-01 0.70455 0.77047 1.00696 0.97947 0.92497 0.94021 MLS001130011-01 0.7512 0.78831 1.0699 0.88339 0.99521 0.95531 MLS001229477-01 0.8339 0.83764 0.90149 0.97022 0.93677 0.80147 MLS000707378-01 1.05105 1.04623 1.24093 0.88992 0.85116 0.87159 MLS000573208-01 0.59664 0.82393 1.04515 0.97999 1.00715 0.94525 MLS001167281-01 0.83453 0.82926 1.14202 1.07436 1.05393 1.06349 MLS000053342-01 1.11028 1.04947 1.03003 1.00173 1.01051 1.05896 MLS002171615-01 0.83027 0.84318 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0.99933 0.95702 MLS000773700-01 0.47196 0.98949 0.95155 0.71177 1.01875 1.00839 MLS001177045-01 0.96779 1.10985 1.0851 1.02356 0.81454 1.04675 MLS000693747-01 1.0456 1.12188 0.91107 1.43271 0.71944 1.06136 MLS001175556-01 0.75145 0.90095 0.90127 1.09994 1.02814 0.94901 MLS001175473-01 0.79712 1.04117 0.99773 0.8872 0.91773 0.95559 MLS002156278-01 1.02456 0.97186 0.99244 0.69084 0.9637 1.00503 MLS000707281-01 1.14087 1.15567 0.75052 1.05935 0.65371 1.01094 MLS000591198-01 0.70144 0.98525 0.85477 1.00046 0.88105 1.01695 MLS000714175-01 1.02244 1.11863 1.01289 1.13111 1.06141 1.08575 MLS002163386-01 1.01995 1.06351 1.06415 1.11206 1.04751 1.10384 MLS000761297-01 1.09327 1.04516 1.03348 1.15364 1.02167 1.0906 MLS002245351-01 1.03102 1.06127 1.042 1.10724 0.94631 1.04658 MLS000718886-01 0.97849 0.93799 0.99814 1.06782 0.95909 0.98188 MLS002156485-01 1.15603 1.05728 1.0687 1.2005 0.98067 1.0551 MLS001140657-01 0.97076 0.9948 0.99812 1.08308 0.95059 1.03407 MLS002157024-01 1.07674 1.18645 0.93233 1.12188 0.88418 1.01348 MLS000721730-01 1.07097 0.87695 1.02458 0.85627 1.06447 0.91949 MLS000705922-01 1.03921 0.90721 0.95439 0.85107 1.13665 0.89838 MLS000724709-01 0.97675 0.885 1.00234 1.04022 1.46739 0.99608 MLS002161757-01 1.14604 0.97951 1.06023 1.09241 1.06958 0.8459 MLS002164687-01 1.0594 1.01681 1.36762 1.23385 1.00235 0.85316 MLS001060533-01 1.11677 1.34224 1.51976 1.31756 1.20754 0.94638 MLS000685139-01 1.26695 1.10202 1.24049 1.12834 0.92023 0.87033 MLS001217286-01 0.5964 1.09526 1.21873 1.19269 0.99106 0.76168 MLS001221619-01 1.03393 0.86027 1.01242 0.94404 0.95568 0.82601 MLS001219621-01 0.45369 0.648 0.86159 0.8883 0.86027 0.9394 MLS001166156-01 0.44609 0.82177 0.75916 1.07035 0.85209 1.00131 MLS000534926-01 0.85648 1.33769 0.9622 1.00584 0.94445 1.02592 MLS000548725-01 0.22187 1.03126 0.84619 0.9206 0.84182 0.86975 MLS000374261-01 0.44896 0.42298 0.92885 1.13089 0.83858 0.81441 MLS000123454-01 0.88519 0.68413 1.08253 1.04655 0.89626 1.06673 MLS000625140-01 0.65137 0.67934 0.91312 1.11353 0.84537 0.84836 MLS001214443-01 1.08326 1.15407 1.19298 0.94422 0.90181 0.82616

TABLE 11 CMV-LACZ in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000591667-01 1.0593 0.8030 1.1592 1.1587 0.7850 0.9491 MLS000568234-01 0.7322 0.8819 0.9894 0.9043 0.9372 0.9556 MLS000689562-01 1.0619 0.9919 1.0858 0.8060 0.7409 0.8453 MLS000732652-01 0.7856 0.9374 1.1235 1.1327 0.7744 0.6700 MLS001240181-01 0.8907 1.0367 1.1645 0.8408 1.0441 0.9550 MLS001211139-01 0.8744 1.0659 0.8608 0.9500 0.7347 0.9691 MLS001030268-01 0.5556 0.7467 1.1787 1.1347 1.1550 1.0809 MLS000912699-01 0.5204 0.8826 1.0462 0.9111 0.8744 0.7147 MLS001125260-01 1.2270 1.0563 0.9131 1.0114 1.0584 1.1581 MLS000717689-01 0.8996 1.0407 1.0936 1.1129 1.0203 1.0125 MLS001197665-01 1.0267 0.9556 0.8403 0.9214 0.8347 0.8584 MLS001075922-01 1.3037 0.9881 1.2361 1.6332 1.3028 0.9434 MLS001124046-01 1.0807 0.9881 0.8501 0.9307 1.2650 0.8409 MLS001197220-01 1.2844 0.9567 1.1383 0.9355 1.1569 1.2028 MLS001221318-01 1.5641 1.0181 0.9955 1.0999 1.0538 0.8528 MLS000947910-01 1.0500 1.0870 1.0379 0.9452 0.9250 1.0094 MLS001215795-01 0.8256 0.8196 0.8628 1.0974 1.1375 0.8003 MLS002163670-01 1.4715 0.8611 0.9725 0.8671 0.8738 1.1806 MLS001200149-01 1.1552 0.9363 1.0523 0.9489 0.9203 1.1234 MLS001359861-01 1.0330 0.9074 0.8064 0.9009 0.9184 0.9444 MLS000710669-01 0.8778 1.1319 1.0823 1.0325 0.9975 1.0969 MLS001035690-01 0.9178 1.3585 1.2020 0.9910 1.0406 0.9819 MLS001030621-01 0.9063 1.0307 1.1844 1.0494 1.0531 0.7081 MLS001083082-01 0.8144 0.9615 1.1713 1.0409 0.9234 0.8591 MLS000045588-01 1.2167 0.8056 1.0611 1.0245 0.8372 0.7647 MLS001216939-01 1.0644 1.1863 0.9452 1.0913 1.1134 1.0991 MLS001163859-01 0.8104 0.7593 1.1812 1.2009 1.1684 1.1113 MLS000683232-01 0.8237 0.6119 0.5224 0.5638 0.7131 0.8043 MLS001170856-01 0.8389 0.8881 1.0004 0.7938 0.9857 0.7337 MLS002667707-01 1.1226 1.0044 1.0803 1.1897 0.6763 1.1434 MLS001200665-01 0.9937 0.7152 1.0036 0.9398 0.9783 1.1043 MLS002161853-01 0.9348 1.0922 0.9436 0.8819 0.8734 1.0131 MLS002163101-01 1.1556 0.9644 1.0313 1.0262 1.0317 0.9991 MLS000062431-01 1.0993 1.0467 1.1632 0.9848 1.1631 0.9557 MLS000028160-01 0.8600 0.8967 1.2759 0.9378 0.9029 1.0663 MLS002248819-01 1.0270 1.7330 1.1575 1.2604 1.2206 0.9206 MLS000080654-01 0.8815 1.0174 0.9345 1.0703 1.4314 1.1851 MLS000760876-01 0.8504 0.9396 0.8973 0.8293 1.0546 0.9069 MLS000677675-01 1.1578 1.0763 1.0373 0.9905 1.1214 1.0343 MLS000113985-01 0.1841 0.2126 0.9187 1.0836 1.0049 0.9809 MLS001182368-01 1.1615 1.2322 0.9225 1.0528 0.8783 0.9571 MLS001212882-01 1.0326 1.3230 1.0086 0.9792 1.1291 1.1409 MLS001004364-01 1.0441 1.0263 0.9011 1.0438 1.0643 0.9117 MLS000736846-01 1.0174 1.3459 1.1101 1.0269 1.0386 1.1009 MLS001098105-01 0.8704 1.0381 0.9421 0.9282 0.8054 1.4689 MLS000678673-01 0.9656 0.8674 0.8991 1.0561 1.1914 1.2106 MLS000925023-01 0.3278 0.5504 0.8397 0.9762 0.9629 1.0703 MLS001212319-01 1.0259 1.2056 0.8600 1.0840 1.2040 0.9129 MLS000779126-01 0.7441 0.9944 0.7610 0.9637 0.9994 0.9751 MLS000948055-01 0.5522 0.5407 1.0993 1.0787 0.9674 0.7540 MLS000110418-01 0.7141 0.8926 0.9455 1.0913 0.9989 0.8871 MLS000693704-01 1.0567 0.7996 0.9977 0.9522 1.1157 0.8989 MLS001225512-01 1.0111 0.8237 1.4266 1.1655 1.0843 0.9597 MLS001006798-01 0.8263 0.9622 1.0189 1.1509 1.0326 0.8334 MLS000711491-01 0.7800 1.5063 1.0199 0.9088 1.1060 1.0889 MLS000582947-01 0.8819 0.7811 1.0067 1.1273 0.8991 0.9691 MLS000531177-01 0.9422 1.2952 0.8958 1.0150 1.5065 1.0785 MLS001202389-01 0.5081 0.6115 0.8920 1.0084 1.1108 1.0237 MLS000536064-01 2.4896 2.5396 1.3388 2.0402 0.7412 0.9105 MLS000586245-01 0.7674 0.7726 1.0505 0.9258 0.8985 0.9034 MLS001061374-01 1.1037 0.9744 0.9793 1.0601 1.1702 0.7117 MLS000675441-01 0.7437 0.8656 0.8690 0.8800 0.9289 1.0332 MLS001200396-01 0.9011 0.7252 1.2107 1.0212 0.9157 1.0489 MLS001165937-01 0.4007 0.4526 0.7441 0.8816 0.7788 0.7295 MLS000325736-01 1.0537 1.0111 1.0696 0.9758 0.9135 0.7862 MLS001215357-01 0.4719 1.2467 1.0378 0.8575 1.2686 1.1560 MLS000588210-01 0.9000 1.0307 0.9316 0.7808 0.9782 1.2908 MLS000764729-01 0.7504 1.1200 0.8880 1.0259 0.9520 0.9865 MLS000689492-01 1.0026 1.0141 0.8653 1.0863 1.0252 0.9363 MLS001000299-01 1.0107 1.1522 0.8248 0.9901 1.2745 1.4188 MLS000393762-01 0.4467 0.3793 1.0198 1.0114 0.9351 0.9560 MLS001130011-01 0.8311 0.7837 1.1138 1.0011 0.7695 1.1702 MLS001229477-01 0.8800 1.0674 1.1311 1.1078 1.1345 1.0283 MLS000707378-01 0.6548 0.8667 0.8072 0.8309 1.0883 1.0175 MLS000573208-01 0.9289 0.6626 0.8678 1.1030 0.9594 1.0197 MLS001167281-01 0.7467 0.9256 1.1951 0.9684 1.1289 1.0114 MLS000053342-01 0.9930 0.9811 0.8785 1.0865 1.0397 1.0289 MLS002171615-01 0.6726 1.2159 0.9905 0.9273 1.3505 1.0498 MLS001005712-01 1.0230 1.0241 0.9125 0.8934 1.2385 1.0188 MLS001176153-01 1.0763 0.9322 0.9140 0.8976 1.4588 1.2729 MLS000735021-01 0.9767 1.2130 0.9907 0.9756 1.1646 0.8683 MLS000767397-01 1.1433 0.8485 0.9687 0.9910 1.0646 1.2748 MLS001196572-01 1.0778 1.0767 0.9456 1.0605 1.3172 1.0105 MLS000393966-01 0.9156 1.0411 0.8087 1.1744 1.0625 0.9249 MLS001034810-01 0.7030 1.1193 0.9459 1.0948 0.9538 0.9698 MLS001165394-01 0.9570 0.8663 0.9792 1.0941 1.0015 1.0655 MLS000089464-01 0.5274 0.7500 1.1969 0.9372 0.9160 0.9117 MLS000698617-01 1.0356 1.1670 1.0603 1.1399 0.9852 1.3409 MLS001175021-01 1.0907 1.1070 0.9395 0.9282 1.0729 1.1772 MLS001166758-01 0.3481 0.6126 1.0661 1.1998 1.0175 0.9357 MLS001008109-01 0.7619 0.8730 1.2178 0.9228 1.1138 0.9542 MLS001181936-01 0.6130 0.7863 0.8296 0.7292 0.9273 0.8487 MLS000560266-01 1.0484 1.1095 0.9991 0.9405 0.8123 0.9030 MLS001215074-01 1.1432 1.1277 1.0294 1.1754 0.7800 0.7877 MLS001215123-01 1.0098 1.2639 1.1943 1.0888 0.8837 1.0530 MLS001033255-01 0.7905 1.1919 1.0219 0.8766 1.0723 0.7590 MLS001160611-01 1.0389 0.8144 1.0340 1.0117 0.8157 1.2070 MLS001006302-01 0.7646 0.9502 1.0767 0.8911 0.7663 0.9343 MLS001123876-01 0.7333 0.9618 0.9871 0.9251 1.0263 0.8260 MLS001122698-01 1.0639 1.0404 0.8608 0.7759 1.1003 1.1513 MLS000755214-01 0.8172 1.0835 1.0994 0.9281 1.1650 1.0190 MLS000731285-01 0.6572 0.9582 0.8436 1.0851 1.0853 1.1747 MLS000776409-01 0.2105 1.3344 0.9052 0.8929 0.9103 1.1220 MLS001221908-01 0.6789 0.9726 0.7908 0.9357 0.7607 0.9663 MLS000419286-01 0.6295 0.9368 0.8022 0.9701 0.7960 0.9960 MLS000554416-01 0.8611 1.1077 1.1123 0.8594 1.3983 1.1310 MLS000073150-01 0.9000 0.9491 0.9954 0.8193 1.8970 1.0753 MLS000663185-01 0.9347 0.9211 0.8567 0.9688 1.1477 1.1390 MLS001078811-01 0.9014 0.8393 0.6873 0.7927 1.2290 1.2397 MLS002694363-01 0.5698 1.0123 1.0392 0.7616 1.2840 1.1110 MLS000689218-01 1.6337 1.2835 1.0813 0.8689 1.2273 1.1977 MLS001215294-01 0.4116 0.3302 1.3412 0.8138 1.2817 1.2983 MLS001183575-01 0.8856 1.2825 0.7762 0.8972 1.2440 1.1603 MLS000393567-01 1.1319 1.0175 0.8148 0.6593 1.1373 1.2117 MLS000546316-01 1.5049 1.3463 1.2440 0.8090 1.1373 1.0623 MLS000912258-01 0.8621 1.1074 1.0171 0.9071 1.1693 1.1073 MLS000850522-01 1.0091 0.8358 0.8671 0.8809 1.3763 0.8880 MLS001197779-01 1.1267 0.9846 0.6490 0.7604 1.2563 0.8987 MLS001095705-01 1.1723 1.1400 0.7229 0.6476 1.2293 1.2060 MLS000912562-01 0.8747 1.0302 0.6246 0.7103 0.9150 0.8680 MLS000092785-01 0.3235 1.1004 0.7351 0.9882 1.1653 0.8600 MLS000418650-01 1.1323 1.1523 0.8344 1.0080 0.9533 0.9237 MLS000860966-01 0.7600 0.6232 0.8424 0.7918 1.0900 0.9370 MLS001180929-01 0.6163 0.9811 0.8753 0.8251 0.9288 0.8571 MLS000564564-01 0.8844 1.0422 0.9091 0.9399 1.0092 0.9117 MLS001164432-01 1.1289 1.1059 1.3183 1.1387 0.7842 0.9600 MLS000621451-01 1.1926 1.2493 1.3023 0.9255 1.1579 1.0388 MLS000078709-01 0.4830 0.6919 1.0163 0.9270 0.7008 0.8167 MLS000688267-01 1.0774 1.1407 0.8935 0.9129 1.1579 1.0950 MLS001006516-01 0.6589 0.4956 0.9076 0.9829 0.8858 1.0038 MLS001095033-01 0.9811 0.9359 1.0269 0.9043 0.9179 0.9488 MLS000072290-01 0.2915 0.3911 1.3303 1.4390 0.9000 1.0579 MLS000036988-01 0.6578 0.8796 1.0435 0.9702 1.0846 1.0767 MLS001217935-01 0.5381 0.7859 1.0465 1.0175 1.1167 1.1729 MLS000777780-01 0.9463 1.3393 1.0126 0.9893 0.9063 0.8779 MLS001174740-01 0.4578 0.4293 0.9490 1.0046 0.9721 1.2750 MLS000392555-01 1.2007 0.9559 0.9799 0.9342 0.7638 0.8954 MLS000693795-01 0.8730 0.8526 0.9207 0.9554 1.1104 0.9100 MLS000684034-01 0.3100 0.5374 1.0568 1.2045 1.1492 1.2525 MLS001172822-01 1.2767 0.9285 1.0904 1.1375 1.0617 0.9304 MLS000052969-01 0.6800 1.1522 1.0444 0.9976 1.0529 0.8950 MLS001217212-01 0.9459 1.1137 1.0626 1.0711 0.9179 1.1804 MLS001004864-01 0.9933 1.2930 1.0417 1.2276 0.9288 1.2650 MLS001116535-01 1.1263 1.1815 1.0580 1.0944 1.1779 1.0167 MLS001165424-01 1.2074 1.2730 1.0078 1.0607 0.9796 1.1754 MLS001116079-01 0.9078 1.0448 1.0228 0.9306 1.0492 1.0950 MLS001198271-01 1.2089 0.9944 0.8516 1.2105 1.0883 1.0204 MLS001167798-01 1.1452 1.2196 1.1494 1.1822 0.7750 1.0342 MLS000710288-01 0.9063 1.1193 1.2515 1.0737 0.9325 1.0338 MLS000734270-01 0.9548 1.0122 0.9468 0.9337 0.8379 0.9296 MLS000858981-01 0.9696 0.9985 0.9009 1.1687 0.9696 0.9229 MLS000698826-01 1.2519 1.2944 0.9013 0.8824 0.9717 0.9942 MLS001000874-01 1.1100 1.0556 1.0295 0.8886 1.0442 1.1029 MLS000682750-01 1.0404 1.1433 0.9934 0.8717 1.0467 0.9679 MLS001090787-01 0.5907 0.7533 0.8594 0.9700 0.9754 0.9196 MLS002636056-01 0.8270 1.1173 1.2571 1.0394 0.9021 0.9392 MLS002170630-01 0.7387 0.8233 0.9628 0.9890 0.8054 0.9954 MLS002162890-01 0.9507 0.7603 0.9939 0.9701 0.8713 0.8392 MLS001105912-01 0.3873 0.5323 0.9597 1.0003 1.0321 1.0725 MLS001007892-01 0.4953 0.6390 1.1495 1.0511 0.8108 0.9338 MLS000089748-01 0.4437 0.3140 0.9905 1.0665 0.8592 1.2129 MLS000912726-01 1.0643 1.0257 1.0325 1.0182 0.9692 1.1004 MLS000086970-01 0.8880 1.0197 0.9068 0.8994 1.0504 0.9925 MLS000420298-01 1.1930 0.9807 1.0482 0.8414 0.9383 1.2808 MLS001147478-01 0.8747 1.0513 1.2999 1.2105 0.8808 1.0721 MLS000090135-01 0.3653 0.6017 0.9296 0.9731 0.9421 1.0896

TABLE 12 CMV1: AB305V-LACZ in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000591667-01 0.8800 0.8929 1.0152 1.0824 1.0726 0.8600 MLS000568234-01 0.9141 0.9918 0.9074 1.0764 1.2622 1.3844 MLS000689562-01 0.9788 0.9824 0.8105 1.0300 0.8652 0.9474 MLS000732652-01 1.0329 0.8518 1.0697 1.0614 0.8859 1.1615 MLS001240181-01 1.1376 1.0976 0.9955 1.0895 1.0511 0.9519 MLS001211139-01 1.0224 0.8259 1.0196 1.1755 0.8948 0.9281 MLS001030268-01 0.9506 0.9365 1.1128 1.1318 1.1711 0.8830 MLS000912699-01 0.5365 0.9235 1.1310 0.9419 0.9859 0.8089 MLS001125260-01 0.9212 1.0000 1.0994 0.9614 1.0993 1.0074 MLS000717689-01 0.9129 0.9576 0.9569 0.9686 1.0956 0.8415 MLS001197665-01 1.0741 1.0047 1.2593 0.9771 0.8756 0.9415 MLS001075922-01 0.8388 1.0282 1.2173 1.2947 1.0533 1.0222 MLS001124046-01 0.9812 1.0906 1.0202 0.8395 1.0156 1.0193 MLS001197220-01 1.0271 0.9659 0.9289 0.9682 0.9356 0.8896 MLS001221318-01 0.8576 1.2212 0.9944 1.1337 1.4059 1.0037 MLS000947910-01 1.0082 1.0165 0.8930 1.2332 0.8348 1.0333 MLS001215795-01 1.1035 1.3929 1.0250 0.9309 1.0207 0.9363 MLS002163670-01 1.1235 1.1659 0.8898 0.9788 1.2044 0.9237 MLS001200149-01 0.9424 0.8318 0.8176 0.9974 0.9933 0.8378 MLS001359861-01 0.9412 0.9976 1.1230 0.9547 0.9533 0.8978 MLS000710669-01 1.1824 0.8682 1.2372 0.9690 0.8881 0.9341 MLS001035690-01 1.0976 1.1976 1.1317 1.1552 0.8644 1.0474 MLS001030621-01 1.0141 0.9024 1.0546 0.9872 0.7970 0.7844 MLS001083082-01 1.1165 1.0282 0.9776 0.9984 1.0326 0.9615 MLS000045588-01 0.9424 1.0494 0.9776 1.2294 0.8607 0.9059 MLS001216939-01 0.9659 1.1518 1.2568 1.1826 0.9748 1.0311 MLS001163859-01 1.2482 1.0965 1.1727 1.0856 1.1326 0.8756 MLS000683232-01 0.8365 0.8835 1.0086 0.8743 0.7752 0.8200 MLS001170856-01 0.8753 1.1518 1.0562 1.0074 0.9105 1.1095 MLS002667707-01 1.1071 1.1576 1.0227 0.9449 1.0590 0.8905 MLS001200665-01 1.2706 1.1800 0.9662 1.1274 0.9695 1.1543 MLS002161853-01 1.0647 1.2341 0.9103 1.1122 0.8352 1.0524 MLS002163101-01 1.5447 0.9212 0.8407 0.8302 1.1400 1.0533 MLS000062431-01 1.2047 0.8812 0.9690 0.9004 0.9743 0.9133 MLS000028160-01 1.1976 1.0471 0.9249 1.0348 1.0362 1.0524 MLS002248819-01 1.1929 0.9847 1.1534 1.1171 0.9505 1.0819 MLS000080654-01 1.4259 1.1871 1.0399 1.0335 1.0086 1.1229 MLS000760876-01 1.2565 0.9482 0.9789 1.1074 1.1010 1.0248 MLS000677675-01 1.2494 0.9918 1.0091 1.0578 0.9962 0.8619 MLS000113985-01 1.0482 1.0541 1.0241 1.0031 1.0638 1.0629 MLS001182368-01 1.0318 0.9953 0.8690 0.8931 1.0619 0.8667 MLS001212882-01 1.3024 1.1471 1.1133 1.0963 0.8705 1.0238 MLS001004364-01 1.1824 1.2824 0.9480 0.8978 1.0095 0.9210 MLS000736846-01 1.2788 1.1000 1.1737 1.0250 0.9714 0.8438 MLS001098105-01 1.2553 0.9671 1.3183 0.9211 1.2543 0.9248 MLS000678673-01 1.1835 0.9635 0.9367 0.7918 1.0286 1.1229 MLS000925023-01 1.1106 1.1565 0.8859 1.1638 1.1333 0.7505 MLS001212319-01 1.0706 0.9965 0.9253 0.9525 0.8152 1.1524 MLS000779126-01 1.1671 0.9988 1.0467 1.0257 1.2162 0.9495 MLS000948055-01 1.0753 1.2212 1.1022 1.2519 0.9638 1.0895 MLS000110418-01 1.3647 1.0459 0.9599 0.9617 0.9000 1.0333 MLS000693704-01 1.0600 1.1647 1.0812 1.1170 0.8695 0.8571 MLS001225512-01 0.8871 1.2482 1.0077 0.9459 1.0381 1.0162 MLS001006798-01 1.0976 0.9953 1.0267 1.2236 0.9648 1.0419 MLS000711491-01 0.9929 1.1318 0.8645 0.9993 0.9095 0.9495 MLS000582947-01 1.0247 1.0729 0.9127 0.9280 0.9610 0.8848 MLS000531177-01 1.1522 0.9244 0.8917 0.8757 1.0411 1.1989 MLS001202389-01 0.9311 1.0900 1.1312 1.0432 1.1821 1.1558 MLS000536064-01 1.0233 1.2344 1.1027 0.8894 1.0389 0.9705 MLS000586245-01 1.2656 1.2267 1.1338 0.9511 1.0800 1.0611 MLS001061374-01 1.0489 1.1689 0.8498 1.1237 1.1000 1.2568 MLS000675441-01 0.9822 0.9578 1.0337 1.0460 1.1032 1.0074 MLS001200396-01 0.9733 0.9644 0.7738 0.8987 0.8442 0.9137 MLS001165937-01 0.7222 0.7678 0.7827 0.8619 0.9663 0.9305 MLS000325736-01 0.9889 0.8244 0.9324 0.9630 0.8968 0.9263 MLS001215357-01 1.0667 1.3622 1.1192 1.1407 1.1400 1.1547 MLS000588210-01 1.1656 1.1322 1.0815 0.9182 1.0853 1.1263 MLS000764729-01 1.1900 0.9656 0.9634 1.0779 1.1432 1.0358 MLS000689492-01 0.9267 1.0444 1.0940 1.0504 1.2400 1.0853 MLS001000299-01 1.0744 0.9544 0.9029 1.0658 1.3158 0.6411 MLS000393762-01 1.0578 1.0800 1.0049 1.0005 0.9326 0.8653 MLS001130011-01 1.0156 0.8367 0.8998 0.9057 1.0211 0.9358 MLS001229477-01 0.9611 0.9622 0.9587 0.9250 1.1095 1.0126 MLS000707378-01 0.9167 0.9689 1.0300 0.9294 1.3937 1.0537 MLS000573208-01 0.9478 0.9633 0.8913 1.1765 1.3021 0.9547 MLS001167281-01 0.9722 1.1089 0.9727 0.8948 1.0789 1.0663 MLS000053342-01 1.1344 1.3044 0.9187 0.9033 0.9863 0.9947 MLS002171615-01 1.0711 1.0111 0.9582 0.9989 0.7874 0.9863 MLS001005712-01 1.3167 1.1133 0.8075 0.9623 1.0379 1.0684 MLS001176153-01 1.0878 1.0256 0.9388 0.7944 0.9832 0.8316 MLS000735021-01 1.0778 1.0378 0.9616 0.9548 1.0221 1.0105 MLS000767397-01 1.0067 1.0144 0.9519 0.9425 1.0368 0.8737 MLS001196572-01 1.1422 1.1456 1.0223 0.9853 0.9379 0.9632 MLS000393966-01 1.1167 1.0378 1.1877 0.8958 1.3516 0.9358 MLS001034810-01 1.1344 1.0578 1.0965 0.9578 1.7389 1.1000 MLS001165394-01 0.9544 1.0344 0.9045 0.9589 1.1558 0.9232 MLS000089464-01 1.0178 1.3067 0.9529 0.9400 1.3716 0.9716 MLS000698617-01 1.0111 1.0011 0.9614 0.9014 1.0842 1.1221 MLS001175021-01 0.9733 1.1378 0.7979 0.9049 0.9621 1.0611 MLS001166758-01 0.9867 1.1044 0.9444 0.9780 0.7474 0.9958 MLS001008109-01 1.0600 1.0756 0.9289 0.9839 1.0168 1.0621 MLS001181936-01 1.0280 0.9020 1.0274 0.9246 0.9886 1.0705 MLS000560266-01 0.9470 1.0520 1.0224 1.0253 1.0000 0.9724 MLS001215074-01 0.9930 0.8700 0.8583 0.8364 0.9238 0.8752 MLS001215123-01 1.1030 1.0710 1.2929 1.1153 1.1457 1.1657 MLS001033255-01 0.7740 0.9930 1.0297 0.9525 1.1438 1.1038 MLS001160611-01 0.9190 1.0240 0.9661 0.8306 1.2419 0.9762 MLS001006302-01 0.9720 0.9010 1.0122 0.9398 0.9143 1.0229 MLS001123876-01 1.0300 0.9880 0.9279 0.9002 1.4943 0.8962 MLS001122698-01 1.0110 1.0340 1.1482 1.2038 1.0133 1.0019 MLS000755214-01 1.0190 0.9280 1.0591 1.0763 0.8600 0.9543 MLS000731285-01 0.6450 1.0310 1.3012 1.4794 1.0562 1.2048 MLS000776409-01 1.1540 1.1040 1.1035 1.0186 0.9752 1.0552 MLS001221908-01 0.8850 1.0770 1.1838 0.9745 0.9171 0.8562 MLS000419286-01 0.7830 0.8530 1.1581 1.0268 1.1867 0.9990 MLS000554416-01 0.7060 0.8760 0.9798 0.8969 0.9629 1.0019 MLS000073150-01 1.0390 0.9750 1.1088 1.2058 1.0848 1.0752 MLS000663185-01 1.1270 0.9540 0.8722 0.9322 0.9800 1.0924 MLS001078811-01 0.6710 1.0510 0.9981 1.1571 0.9086 0.8486 MLS002694363-01 0.7460 0.9870 0.9775 1.1420 1.1800 1.0695 MLS000689218-01 1.7990 1.4900 1.2135 1.3684 0.9514 0.9657 MLS001215294-01 1.4470 1.2800 1.4324 1.1438 1.0800 0.8838 MLS001183575-01 1.1230 0.8230 0.9136 0.7726 0.8657 0.9648 MLS000393567-01 0.9100 1.0160 0.9858 0.9322 0.9524 1.0400 MLS000546316-01 1.2640 1.0190 1.0825 1.0040 1.0019 1.2590 MLS000912258-01 1.0350 1.0760 1.2253 1.1251 0.9162 0.9743 MLS000850522-01 1.0200 1.0580 1.0751 1.1090 0.8933 0.8848 MLS001197779-01 1.0540 1.0630 1.0983 1.0456 1.1705 1.1286 MLS001095705-01 1.0800 1.2020 1.3164 1.1898 0.9933 0.9486 MLS000912562-01 1.2780 1.2090 1.1674 0.9602 0.8924 1.0905 MLS000092785-01 1.0500 1.0400 1.0738 1.0185 1.0143 0.9105 MLS000418650-01 1.2260 1.1180 1.1746 1.0198 1.2819 1.0086 MLS000860966-01 0.7920 0.9190 0.8601 0.9632 1.0743 0.8229 MLS001180929-01 1.0274 1.1347 1.0084 1.0533 0.7876 0.8848 MLS000564564-01 1.0411 0.9779 1.1848 1.0689 0.9924 0.8267 MLS001164432-01 1.1179 1.1358 1.2418 0.9500 1.3543 0.9248 MLS000621451-01 0.9695 0.9853 1.1398 1.0177 0.9295 1.1114 MLS000078709-01 1.7737 1.0853 0.8743 0.9348 0.9257 1.0133 MLS000688267-01 1.1505 0.9505 1.2248 0.9634 1.1333 0.8648 MLS001006516-01 0.9484 0.9032 0.8542 0.9283 0.9257 0.9581 MLS001095033-01 1.0358 1.0326 1.0031 1.0230 1.0867 1.2895 MLS000072290-01 1.0653 1.3484 1.1459 1.3265 1.1495 0.9362 MLS000036988-01 0.5021 0.7116 1.0868 0.8979 0.8905 0.9867 MLS001217935-01 0.9495 1.0547 0.9648 1.1473 1.1381 0.9705 MLS000777780-01 1.0474 0.9316 0.8808 1.0471 0.8838 0.8914 MLS001174740-01 0.8863 1.0568 1.0520 0.9101 0.9267 0.8533 MLS000392555-01 0.9326 1.1537 0.8568 1.0094 0.9676 1.0257 MLS000693795-01 1.0716 1.0453 1.0774 1.1780 1.0314 0.9771 MLS000684034-01 1.0105 1.2347 1.1903 1.1407 0.9810 1.0962 MLS001172822-01 0.6358 1.1768 1.1819 1.2505 1.0514 1.2590 MLS000052969-01 0.9726 0.9800 0.8406 1.0651 1.1714 0.9152 MLS001217212-01 1.0105 1.1842 1.0832 0.9795 0.9610 1.0190 MLS001004864-01 0.8884 0.9653 0.9501 1.0697 0.9610 0.9657 MLS001116535-01 1.0011 0.9800 1.2856 1.0648 0.8771 1.2552 MLS001165424-01 0.8558 1.0316 1.1758 1.0571 1.1248 0.9152 MLS001116079-01 1.0811 0.9063 1.1374 1.0227 0.8152 1.3543 MLS001198271-01 1.0053 1.1053 0.9957 0.9907 1.0686 0.9933 MLS001167798-01 1.2800 1.3337 1.2238 1.1987 0.9295 0.9143 MLS000710288-01 0.8905 0.9400 1.1225 1.0717 1.1133 1.0371 MLS000734270-01 0.9095 1.1158 1.0885 1.1042 1.0038 1.0019 MLS000858981-01 1.0000 1.0421 0.9781 1.0786 0.8419 1.0257 MLS000698826-01 1.0789 1.0505 0.9634 1.1232 1.0200 1.2010 MLS001000874-01 1.0947 1.1484 0.9960 1.1124 1.1590 1.0943 MLS000682750-01 1.0579 1.1189 1.0939 1.1285 0.9638 1.0248 MLS001090787-01 0.9450 0.8990 1.1178 1.0126 0.9055 1.0009 MLS002636056-01 1.1160 1.4090 1.1613 1.0269 1.2636 0.9855 MLS002170630-01 0.8580 0.9710 0.9248 1.2068 1.0036 0.8136 MLS002162890-01 1.0170 1.0140 1.0530 1.1237 1.0191 1.0682 MLS001105912-01 1.8130 1.1910 1.0857 0.9313 0.9618 1.1391 MLS001007892-01 1.0540 1.2170 0.9543 1.0066 0.9291 1.1673 MLS000089748-01 1.2820 1.3800 0.9813 1.1104 0.9809 0.9527 MLS000912726-01 1.0150 1.2060 1.1041 0.9111 0.9264 0.8527 MLS000086970-01 0.9880 0.9870 0.9738 1.1136 0.8855 0.9673 MLS000420298-01 1.1250 1.3660 1.2388 1.0338 0.8527 0.9573 MLS001147478-01 0.9510 1.0810 1.2959 0.8937 1.3827 1.3536 MLS000090135-01 1.1840 1.1100 1.0114 0.7503 1.1036 1.0155 MLS001179717-01 0.48286 0.57476 0.81333 1.084 MLS000683234-01 0.93 0.69524 0.81111 0.82044 MLS000695955-01 0.93 0.99238 0.896 0.78933 MLS001125488-01 0.87238 1.1819 0.96756 0.74711 MLS000768008-01 0.94286 1.02762 0.96444 0.82756 MLS000913117-01 0.65381 0.59381 0.82622 0.80356 MLS000860538-01 0.96667 1.01667 0.96133 1.00133 MLS001177259-01 1.10048 0.85429 0.73156 0.94622 MLS000861434-01 0.8381 0.84286 0.80267 0.88444 MLS000047918-01 1.05 1.03762 0.8 0.93733 MLS000389484-01 2.78095 1.11571 0.83867 0.85244 MLS001217673-01 0.93524 1.16095 0.94089 0.936 MLS000389677-01 0.76095 1.01857 0.74178 0.81422 MLS001208858-01 0.92381 0.94714 0.80178 0.944 MLS000333610-01 0.99476 0.94286 0.78667 0.832 MLS001117351-01 0.89524 1.1481 1.08756 1.01644 MLS000682883-01 0.89667 1.08667 1.08356 0.93333 MLS001095231-01 0.62286 0.9181 0.74089 1.03511 MLS000721584-01 1.1019 1.13333 0.90711 0.95733 MLS001183429-01 0.58095 0.91048 0.944 0.84356 MLS002158881-01 0.80952 0.86429 0.82667 0.79244 MLS001165749-01 1.03762 1.02429 0.92844 0.82711 MLS001237320-01 1.08238 0.98952 0.75689 0.95822 MLS000763405-01 0.80238 0.82429 0.876 1.04978 MLS000538580-01 0.27048 0.38381 0.57289 0.66133 MLS001033202-01 1.19952 0.82143 0.99333 0.96578 MLS001216260-01 0.83667 1.08952 1.03111 0.85156 MLS000085522-01 0.95095 0.77333 0.98044 1.092 MLS000702680-01 1.18143 1.23286 1.10711 1.00756 MLS001212998-01 0.78333 0.9719 0.90844 0.78 MLS001160885-01 0.8319 0.90952 0.90089 1.16711 MLS001122718-01 0.89 0.95667 0.96622 0.88622 MLS000027478-01 0.97905 0.94429 0.924 0.94889 MLS001177364-01 0.97429 1.05286 0.95778 1.26222 MLS001179695-01 1.04714 1.17524 1.09467 1.18311 MLS002251986-01 0.97762 0.82381 0.96089 0.98311 MLS001166704-01 1.0685 1.4595 1.1553 1.08848 MLS001196422-01 0.856 0.8295 0.89309 0.89631 MLS001179624-01 0.4965 0.452 0.8788 0.86037 MLS001223425-01 1.0095 0.979 1.03502 0.95622 MLS001117140-01 1.1745 1.3785 1.12442 0.95069 MLS001110618-01 0.4765 1.0555 0.93364 0.89539 MLS001223482-01 0.8095 0.858 0.79309 0.90691 MLS000680049-01 0.4315 1.4405 1.10599 1.12719 MLS001212498-01 0.8475 0.749 0.78387 0.84977 MLS001124732-01 0.972 1.07 1.01567 1.02488 MLS000526364-01 1.1865 1.1735 0.953 0.95069 MLS000767227-01 1.1815 0.9975 1.09954 1.02396 MLS000703499-01 0.769 0.689 0.99677 0.93134 MLS001167169-01 1.103 1.085 1.00876 0.93226 MLS001198693-01 1.112 1.025 0.92581 1.09171 MLS001219345-01 1.0255 1.0075 0.86313 1.18802 MLS001211651-01 0.977 0.8465 0.84332 0.87143 MLS000806880-01 1.0655 0.9145 1.12995 0.96544 MLS001223567-01 1.012 1.241 0.89309 1.00184 MLS001005283-01 1.1425 1.032 0.90507 0.90046 MLS001218427-01 0.6965 34.571 15.1793 1.20922 MLS001139288-01 1.0825 1.096 1.04654 1.24654 MLS000696445-01 0.924 1.122 1.01106 1.26774 MLS001218795-01 0.989 1.2115 1.02258 0.9553 MLS000419555-01 1.22 1.2165 1.21475 1.21982 MLS001225507-01 0.8805 0.8655 0.99401 1.04009 MLS000663651-01 0.861 0.949 0.9788 0.96359 MLS000706349-01 1.1355 1.0245 0.9553 0.94654 MLS000393110-01 0.8895 1.0745 0.96728 1.10323 MLS000574647-01 1.0095 0.962 0.93641 0.91659 MLS000532969-01 0.8385 0.8525 0.88295 0.77051 MLS001125260-01 1.175 1.0765 1.06498 0.96452 MLS000122749-01 1.0025 0.808 1.06267 1.02166 MLS001150751-01 0.889 0.992 0.83364 0.91336 MLS001221867-01 1.0745 1.0515 0.98341 0.99539 MLS001147727-01 1.1835 1.2365 0.90691 1.11982 MLS000688437-01 1.05088 1.02982 0.93462 1.23248 MLS001211976-01 1.01404 0.96974 0.99786 0.8547 MLS002161350-01 1.04561 0.88465 0.96795 0.90598 MLS001077207-01 0.78246 0.86623 1.0359 0.95214 MLS001209245-01 0.99518 0.96623 0.96752 0.85684 MLS000737953-01 0.90965 0.94781 1.22393 0.82735 MLS000552080-01 0.80439 0.95219 1.0688 1.19658 MLS000737204-01 0.3807 0.57719 0.80684 1.02906 MLS000579238-01 0.9443 0.83026 0.87179 0.81453 MLS001181671-01 0.93772 1.03026 0.92009 0.88034 MLS001167424-01 0.99254 1.15702 0.97179 0.74915 MLS000094770-01 0.84211 0.94123 0.9406 0.80812 MLS001123810-01 0.93465 1.15921 0.81496 0.8812 MLS000532078-01 0.86228 0.9443 0.89145 0.79103 MLS000585616-01 0.87939 0.90965 0.7906 0.90256 MLS000553673-01 0.91272 1.00526 0.96026 0.80214 MLS001175592-01 1.01842 0.96491 1.03077 0.94701 MLS001033255-01 1.23553 1.01272 1.04487 0.84231 MLS000733703-01 0.79649 0.95833 0.86538 1.1188 MLS001096269-01 0.77544 0.9864 0.74017 1.08974 MLS001162872-01 0.97939 1.06096 1.08376 0.78547 MLS000584511-01 0.74781 0.87105 0.8735 0.83547 MLS001166325-01 0.87851 0.89693 0.97949 0.77265 MLS000122180-01 1.00702 0.8136 0.88077 0.77735 MLS001157804-01 1.06974 1.05044 0.98419 0.70299 MLS000693729-01 0.87895 0.90307 1.05684 0.97222 MLS001220669-01 1.00044 1.0114 0.77009 0.82735 MLS001214461-01 0.95658 0.91667 0.93632 0.81368 MLS000081838-01 0.96974 1.01842 0.88889 0.91752 MLS002402866-01 0.7307 0.83202 0.79658 0.87393 MLS001200980-01 1.0886 0.92719 0.95684 0.87821 MLS001174719-01 1.10789 0.92544 0.83205 0.85513 MLS001175449-01 0.86711 0.88904 0.90342 0.85342 MLS001172577-01 0.96272 1.02895 0.86197 0.89444 MLS001122792-01 0.72544 0.99693 0.96752 0.81368 MLS001216405-01 1.07982 1.34474 0.80085 0.91923 MLS000673766-01 1.04045 1.10955 0.96316 0.98469 MLS000912614-01 1.01227 0.97 1.16603 1.01818 MLS001194551-01 0.78091 1.04682 0.94593 0.82727 MLS001214704-01 0.91364 0.93273 1.27847 0.88517 MLS001219159-01 1.02318 0.96364 0.93158 0.93541 MLS001060549-01 1.075 0.88136 0.89522 1.32153 MLS000850824-01 0.94591 0.99455 0.99234 0.88517 MLS000879190-01 0.46273 0.84955 1.04737 0.99091 MLS001163140-01 1.18955 0.91591 1.06124 1.02871 MLS000046123-01 1.07636 1.14455 1.08421 0.91244 MLS000086970-01 0.82727 0.93955 1.18565 0.95407 MLS000388722-01 0.86682 1.08455 1.06699 0.95694 MLS000676974-01 0.46 1.08273 0.45742 1.07656 MLS000772580-01 1.08773 1.02909 1.0933 0.89856 MLS000698686-01 0.88636 0.88636 0.97273 0.83636 MLS001162337-01 1.05 0.84909 0.95933 0.99043 MLS001204005-01 0.78136 1.01818 1.13636 0.94019 MLS000721525-01 0.91273 0.84091 1.03636 0.86603 MLS000525404-01 0.74955 0.84136 0.87273 0.76029 MLS000772194-01 0.99682 1.05636 0.92344 1.06842 MLS000775793-01 1.02136 0.93227 1.01196 0.85502 MLS000710130-01 0.93364 0.98636 0.86459 0.81435 MLS001146463-01 1.14091 1.06045 0.93923 1.07416 MLS000712769-01 0.85818 0.91409 1.07129 0.96507 MLS000334464-01 0.96364 0.98773 1.07751 1.30144 MLS000862690-01 1.01818 0.93227 0.89426 0.86842 MLS001179267-01 0.55545 0.61409 0.8244 0.90909 MLS000683174-01 1.23773 1.035 1.0866 1.15646 MLS000913052-01 0.92 0.91545 1.28708 1.11531 MLS001080869-01 1.01045 0.99273 0.91531 0.93493 MLS000332693-01 0.93045 0.88045 0.97943 1.24402 MLS001141113-01 1.91273 0.98227 1.38278 1.13397 MLS001176611-01 0.785 1.03364 1.23541 0.92871 MLS001202627-01 0.735 0.93455 0.9555 0.92344 MLS000765108-01 0.97 0.99273 1.09856 1.17656 MLS000937079-01 0.96818 0.96409 0.99234 1.08708 MLS001215742-01 0.98509 0.92544 1.1034 1.02596 MLS001217045-01 0.91272 0.92719 0.98213 0.96128 MLS001196946-01 3.9614 2.7886 1.12979 0.9817 MLS001216714-01 3.63333 1.41711 0.98 0.91617 MLS000772430-01 1.03684 0.89561 0.90936 0.8183 MLS000693370-01 1.1136 0.99474 0.96213 0.9783 MLS000769322-01 1.09079 1.11842 1.01957 0.86085 MLS000721030-01 0.79167 0.92763 0.96553 1.11106 MLS001176897-01 1.04649 1.12149 0.79702 1.00851 MLS000774940-01 1.00526 1.17105 0.86043 0.95404 MLS001030746-01 0.99123 1.09781 0.87574 0.97915 MLS003126425-01 0.71228 0.74298 0.89021 1.35447 MLS001217697-01 0.73947 0.76404 0.86468 1.20043 MLS000516719-01 1.07193 1.10307 1.11404 0.88936 MLS001165323-01 1.48772 1.31535 0.9 1.07234 MLS001220803-01 1.04956 1.09079 1.06426 1.11489 MLS001163121-01 0.92105 0.87807 0.82936 0.85064 MLS001060561-01 0.64254 0.93728 1.00213 0.93957 MLS001139515-01 0.96009 0.92368 0.76043 0.94043 MLS001149811-01 1.65044 1.12193 1.04766 0.9566 MLS000773700-01 0.40921 0.92895 1.1166 1.12553 MLS001177045-01 1.11798 1.05833 1.11021 0.92383 MLS000693747-01 1.04298 0.9693 0.95234 0.99617 MLS001175556-01 1.10263 0.91579 1.05277 1.11277 MLS001175473-01 1.06579 0.95921 0.85872 0.89617 MLS002156278-01 1.03114 0.96667 1.09574 0.86936 MLS000707281-01 0.95263 0.94737 0.92553 0.87064 MLS000591198-01 0.45307 0.83377 0.93191 0.96809 MLS000714175-01 0.97105 1.12412 1.09745 0.93191 MLS002163386-01 1.06623 0.94649 1.19617 0.97489 MLS000761297-01 0.88289 0.99298 0.81064 0.99106 MLS002245351-01 0.9307 1.00921 1.00128 0.83064 MLS000718886-01 0.83553 0.87982 0.98043 0.97872 MLS002156485-01 0.94825 1.11491 0.95234 0.94681 MLS001140657-01 1.075 1.05044 1.04596 1.05319 MLS002157024-01 0.83333 1.05921 0.85532 1.14638 MLS000721730-01 1.03067 0.93111 0.92069 0.85043 MLS000705922-01 0.99467 0.93022 0.9306 0.87759 MLS000724709-01 1.12533 1.05378 0.88276 0.84224 MLS002161757-01 1.256 1.15289 1.10216 0.91897 MLS002164687-01 1.11333 0.968 0.92198 0.7625 MLS001060533-01 0.68711 0.77156 0.77888 0.83578 MLS000685139-01 0.93867 1.03111 1.03621 0.93621 MLS001217286-01 0.36044 0.39911 1.22672 0.95991 MLS001221619-01 1.78133 1.51333 1.32069 1.17155 MLS001219621-01 1.29022 1.132 1.06293 0.94914 MLS001166156-01 0.52267 0.77956 0.91983 0.87069 MLS000534926-01 0.844 0.95467 0.96897 0.97241 MLS000548725-01 0.332 0.87289 1.12629 0.97888 MLS000374261-01 0.50356 0.448 0.8319 0.82457 MLS000123454-01 1.05467 0.87956 0.93233 0.93534 MLS000625140-01 1.016 1.10667 1.03621 0.91724 MLS001214443-01 0.90222 0.95067 1.07241 0.97371

TABLE 13 Burkin Assay in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000763074-01 0.60851 0.65857 1.13427 0.843 1.06668 1.19903 MLS000564846-01 0.75904 0.74063 0.87038 0.80671 0.84376 0.83593 MLS001202366-01 0.24561 0.48734 0.74576 0.87622 0.96981 1.00673 MLS001182278-01 0.84246 0.76628 1.17046 1.06287 1.0742 1.21321 MLS001196427-01 0.87626 0.81017 0.97767 0.93838 1.03303 1.01962 MLS001202425-01 0.34448 0.74647 0.86448 0.96729 1.21932 1.2214 MLS001214276-01 0.02694 0.20219 0.32354 0.62344 1.22246 1.32758 MLS000682748-01 0.24723 0.46691 0.765 0.89229 1.16212 1.259 MLS000834756-01 1.08092 1.06424 1.17192 1.22657 1.09843 1.05646 MLS001202402-01 0.32496 0.64813 1.08015 0.92555 0.90338 1.05088 MLS001214264-01 0.23942 0.78788 0.70087 0.74298 0.84644 0.8908 MLS000391588-01 0.30047 0.78036 0.81758 0.91191 1.01537 1.06962 MLS001013431-01 0.33625 0.45216 0.53107 0.70768 1.00039 1.20211 MLS001163848-01 0.14441 0.29703 0.31406 0.48542 0.89062 1.00231 MLS000327715-01 0.57757 0.90154 1.02899 1.14772 1.09422 1.37572 MLS001214300-01 0.02593 0.18472 0.35298 0.80412 0.93656 1.21103 MLS000834755-01 0.79619 1.19253 1.15647 1.08036 1.07782 1.27365 MLS001163860-01 0.23471 0.17222 0.49218 0.57932 0.84566 0.94942 MLS000710233-01 0.17433 0.17232 0.56369 0.80665 1.15918 1.17028 MLS002702497-01 0.04886 0.05588 0.18885 0.17496 0.55454 0.78041 MLS000546982-01 0.88835 1.05267 1.07821 1.27207 1.15498 0.87993 MLS001197838-01 0.86478 0.89105 1.04838 1.05318 1.19792 1.25617 MLS001202354-01 0.42755 0.51598 1.07323 1.03576 1.02304 1.07717 MLS001202330-01 0.64675 0.83048 0.93405 0.95578 1.02782 0.97384 MLS001182377-01 0.61764 0.87441 0.90613 0.91503 1.04505 1.17882 MLS000834757-01 1.02067 1.11712 0.96506 1.12452 1.00317 1.0102 MLS001202634-01 0.93072 0.99395 1.31203 1.14099 1.10045 1.20777 MLS001194544-01 0.50277 0.79143 1.14522 1.02036 0.99063 1.04429 MLS000554109-01 0.89366 0.9611 1.09535 1.06742 1.04915 0.99289 NCGC00263072-01 0.98868 1.10983 1.04886 1.01335 1.24172 1.27253 NCGC00263071-01 0.53023 0.69803 0.78353 0.9185 1.13222 1.35429 NCGC00263039-01 0.81077 0.99561 1.02563 1.02082 0.91594 1.1697 NCGC00164631-03 0.01283 0.01408 0.01373 0.32241 1.03203 0.83726 NCGC00179302-02 0.80668 0.84305 0.71633 0.60599 1.0859 1.06101 NCGC00241113-01 0.9168 0.95936 0.86618 0.9062 1.05675 1.01086 NCGC00094381-04 0.64658 0.77957 0.63974 0.7317 1.05336 1.03555 NCGC00015546-04 0.14966 0.19368 0.21399 0.37707 0.92282 0.89229 NCGC00094381-03 0.52027 0.6668 0.65793 0.73271 0.9952 0.93685 NCGC00094381-05 0.75776 0.88787 0.7145 0.75321 0.98039 0.94327 NCGC00015545-02 0.18236 0.30858 0.31898 0.59598 1.32546 1.17517 NCGC00164631-04 0.01361 0.01476 0.08696 0.52898 1.01135 1.02287 NCGC00094112-04 0.34285 0.45094 0.49426 0.57218 0.99267 1.01949 NCGC00242500-01 0.01945 0.01841 0.58791 0.87045 1.07607 1.05199 NCGC00015545-07 0.242 0.30684 0.56798 0.84629 1.12041 1.07252

TABLE 14 Burkin Assay in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000763074-01 0.26221 0.19215 0.90703 1.04266 1.12824 1.0274 MLS000564846-01 0.75294 0.70065 1.07066 1.00195 0.95569 0.88356 MLS001202366-01 0.51046 0.52721 0.91937 1.08888 1.09876 1.11392 MLS001182278-01 0.64876 0.56532 1.41234 1.40178 1.11835 1.1969 MLS001196427-01 0.93648 0.79418 1.13545 1.02154 1.01999 0.91943 MLS001202425-01 0.35401 0.51782 0.80333 0.9499 1.20487 1.14828 MLS001214276-01 0.07736 0.23625 0.69453 0.9019 1.19651 1.16185 MLS000682748-01 0.62062 0.74301 1.08485 1.14662 1.21063 0.98015 MLS000834756-01 0.91886 0.94946 0.9214 1.09476 1.18855 1.07853 MLS001202402-01 0.37173 0.59069 1.0295 0.96293 0.89618 0.96279 MLS001214264-01 0.71731 0.71648 1.05756 0.90726 0.89746 0.95761 MLS000391588-01 0.66403 0.76307 0.93558 0.89942 0.95626 1.07272 MLS001013431-01 0.70692 0.7376 0.85055 0.85067 1.0097 1.23758 MLS001163848-01 0.48759 0.68784 0.74688 0.95866 1.04499 1.0964 MLS000327715-01 1.11892 1.19159 1.11494 1.30229 1.02522 0.88377 MLS001214300-01 0.31784 0.74468 0.87335 1.09139 1.03917 1.01853 MLS000834755-01 0.81603 0.96865 0.98675 1.00334 1.23201 1.26329 MLS001163860-01 1.20854 0.86198 1.13792 0.92233 0.94035 1.00928 MLS000710233-01 0.62947 0.69332 0.86883 0.93307 1.033 1.11028 MLS002702497-01 0.38389 0.45669 0.78137 0.76924 0.74901 0.84142 MLS000546982-01 0.71115 0.85558 0.99622 1.14451 1.03799 1.04819 MLS001197838-01 0.99815 0.86987 1.09145 1.00443 0.98105 1.00877 MLS001202354-01 0.57849 0.5824 1.06858 0.95451 0.90225 0.84594 MLS001202330-01 0.62071 0.63028 1.01709 0.91234 0.91736 0.97095 MLS001182377-01 0.91488 0.86542 0.9389 0.93717 1.02095 1.01535 MLS000834757-01 0.94642 0.99887 0.95369 1.02962 0.94297 1.01879 MLS001202634-01 1.28943 0.94727 1.13854 0.9675 1.1156 0.99278 MLS001194544-01 0.76105 0.754 1.06983 1.00203 0.9995 0.96112 MLS000554109-01 1.04819 0.84256 0.98744 0.96115 1.05362 0.88389 NCGC00263072-01 0.88975 0.93047 0.95374 0.96945 1.00581 0.98195 NCGC00263071-01 0.65683 0.68367 0.77685 0.86036 0.89218 0.86806 NCGC00263039-01 0.59697 0.74735 0.86326 0.98314 0.95574 0.96891 NCGC00164631-03 0.07758 0.09031 0.18808 0.55055 0.94454 0.95119 NCGC00179302-02 0.68712 0.68199 0.79125 0.81812 0.93769 0.93491 NCGC00241113-01 0.63117 0.74575 0.76777 0.84894 1.10523 1.07764 NCGC00094381-04 0.93082 0.76798 0.87417 0.83691 0.94176 0.8006 NCGC00015546-04 0.79646 0.78811 0.82527 0.85291 1.05081 0.9443 NCGC00094381-03 1.15097 0.94969 1.02446 0.90148 0.93337 0.8564 NCGC00094381-05 1.13784 1.05028 1.31892 1.04722 0.93095 0.92337 NCGC00015545-02 0.35192 0.49594 0.55162 0.69213 0.91177 0.91301 NCGC00164631-04 0.08093 0.08218 0.21321 0.77386 0.99976 0.97606 NCGC00094112-04 0.34997 0.68129 0.62896 0.7596 0.81588 0.74019 NCGC00242500-01 0.21319 0.652 0.75336 0.94215 0.93914 0.99743 NCGC00015545-07 0.49755 0.66539 0.74573 0.87208 1.08023 1.12579

TABLE 15 CMV-LACZ in Myoblasts 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000763074-01 1.6644 1.2981 1.1966 1.1447 0.8256 0.8431 MLS000564846-01 1.0885 1.2363 1.3619 0.9379 0.8172 0.9922 MLS001202366-01 0.3322 0.7222 1.3214 1.0533 0.6431 0.7656 MLS001182278-01 0.5959 1.0519 0.9778 0.9823 0.9491 0.7466 MLS001196427-01 0.9563 0.7144 1.1811 0.9957 1.0356 0.7219 MLS001202425-01 0.6233 0.9081 1.1386 0.8675 1.0919 1.0625 MLS001214276-01 0.1781 1.2922 1.2231 1.3014 0.8731 0.9516 MLS000682748-01 0.2419 0.3893 0.9959 1.0215 0.9666 0.9075 MLS000834756-01 1.1889 0.8022 1.0917 1.2652 1.0422 0.8709 MLS001202402-01 0.4819 0.7204 0.7598 0.8771 1.0106 1.0546 MLS001214264-01 0.4856 0.3870 0.9233 1.3058 1.1383 1.0657 MLS000391588-01 1.1119 1.4307 1.0968 0.9885 0.7411 1.0009 MLS001013431-01 0.6670 0.5904 0.6673 0.8366 1.0640 0.9906 MLS001163848-01 2.2215 2.1030 1.0762 0.9336 0.9620 1.1146 MLS000327715-01 1.0648 1.3181 1.0060 0.9664 1.1731 1.2294 MLS001214300-01 0.1793 0.4007 1.1047 0.7949 0.9403 1.0446 MLS000834755-01 0.9133 0.9007 0.9834 0.9591 1.0400 1.0317 MLS001163860-01 0.6488 0.3425 1.1417 0.9610 0.8230 0.9350 MLS000710233-01 0.8779 0.8530 0.8509 0.8263 0.8263 1.0173 MLS002702497-01 0.2779 0.2593 3.6076 2.6344 1.0213 0.8643 MLS000546982-01 1.3937 0.9512 1.0785 0.9273 1.2640 1.0877 MLS001197838-01 0.9437 1.1615 1.1118 1.0010 0.9954 0.7904 MLS001202354-01 0.7437 0.8378 0.9170 0.9669 0.9521 1.2275 MLS001202330-01 0.7619 0.9511 0.9082 1.0099 0.9325 0.7417 MLS001182377-01 0.4170 0.4441 0.9294 0.9113 1.1646 1.1229 MLS000834757-01 1.1026 0.8115 1.0942 0.9101 0.9300 1.0367 MLS001202634-01 0.9217 1.0817 1.0990 1.1287 1.0071 1.0500 MLS001194544-01 0.7383 0.8983 1.0363 0.9068 0.7213 0.8975 MLS000554109-01 0.9650 0.9853 0.9457 0.9003 1.1863 1.1083 NCGC00263072-01 1.2880 1.1540 1.1158 1.0035 0.9983 1.2383 NCGC00263071-01 0.6430 0.7803 0.9908 1.2012 1.2813 1.0800 NCGC00263039-01 0.9543 0.9740 0.9335 1.0522 1.1700 1.0746 NCGC00164631-03 0.1767 0.1830 0.2763 0.7817 0.8658 NCGC00179302-02 1.2873 1.0247 1.6628 2.4679 1.1867 NCGC00241113-01 3.3097 2.7827 1.1352 1.1634 1.4142 NCGC00094381-04 0.9697 0.9330 0.9770 1.1366 1.0763 NCGC00015546-04 0.5873 0.8083 1.2439 1.4889 1.1442 NCGC00094381-03 1.0253 0.7777 1.0089 0.8667 0.9504 NCGC00094381-05 0.8220 0.7147 0.8606 0.9614 0.8383 NCGC00015545-02 0.2480 0.2907 0.8475 0.9440 1.0179 NCGC00164631-04 0.1827 0.2323 0.4405 0.6842 0.8225 NCGC00094112-04 0.3627 0.4403 1.0782 1.0309 1.1779 1.1721 NCGC00242500-01 0.4237 0.3570 2.4909 1.6977 1.1446 1.1021 NCGC00015545-07 0.3383 0.5257 0.8603 0.7896 1.1950 1.2967

TABLE 16 CMV-LACZ in Myotubes 40 uM 20 uM 10 uM 5 uM 1 uM 0.5 uM MLS000763074-01 1.9682 1.7671 0.9645 1.0487 0.8593 1.0089 MLS000564846-01 1.0094 1.2012 0.8711 1.1234 1.0444 0.8304 MLS001202366-01 1.0847 0.8329 0.9927 0.8072 1.0570 0.9844 MLS001182278-01 1.1212 1.0224 1.0297 1.0327 1.0126 0.9674 MLS001196427-01 0.9000 0.9918 0.9282 0.8423 1.0704 0.9363 MLS001202425-01 0.9976 0.9918 1.0423 1.1496 1.0185 0.8793 MLS001214276-01 0.4976 0.5259 11.1000 2.0151 0.9141 0.7733 MLS000682748-01 1.1071 1.1753 0.9205 0.8790 0.8459 0.9356 MLS000834756-01 2.2953 2.0788 1.1713 1.1994 1.0059 0.9859 MLS001202402-01 0.8129 0.8847 0.8015 0.9237 0.8695 0.9200 MLS001214264-01 1.4482 1.2424 1.1534 0.9411 1.1905 1.1210 MLS000391588-01 0.9094 0.9212 0.9740 1.0019 1.0010 0.8400 MLS001013431-01 1.2059 1.0682 0.9162 0.9388 1.1257 0.8638 MLS001163848-01 6.8624 6.7729 4.2586 1.1937 0.9638 1.2133 MLS000327715-01 1.4871 1.2200 0.9983 1.1289 1.0562 1.1762 MLS001214300-01 0.7118 6.2635 1.0956 1.0550 0.8924 0.9819 MLS000834755-01 0.8789 1.2989 1.1048 1.0514 1.2863 0.5547 MLS001163860-01 1.4490 1.3880 1.2757 1.1250 1.1562 1.0352 MLS000710233-01 1.1380 1.1130 1.0698 0.9060 0.9410 0.8781 MLS002702497-01 0.6550 0.6520 6.3598 7.0726 2.2829 1.1181 MLS000546982-01 1.3290 1.0160 1.1265 1.0159 0.8467 1.0438 MLS001197838-01 1.0063 0.8547 1.1637 0.9701 1.0038 1.0305 MLS001202354-01 0.7863 0.7000 1.0340 0.8910 0.9200 1.0676 MLS001202330-01 0.8242 0.9168 0.8717 0.9600 0.9562 0.8314 MLS001182377-01 1.0263 1.0305 1.0589 0.9608 0.8105 0.7762 MLS000834757-01 1.3600 1.0789 1.1047 1.0546 0.8695 1.0238 MLS001202634-01 0.9340 0.9750 0.9661 1.2348 1.0073 0.9582 MLS001194544-01 0.9400 0.9800 0.9739 0.9683 1.1318 1.0764 MLS000554109-01 0.9950 0.9100 1.1355 1.1559 0.9491 1.2682 NCGC00263072-01 1.0230 0.9190 1.0660 1.0396 0.8536 1.0945 NCGC00263071-01 0.9160 1.0840 0.9779 1.0165 1.2909 0.9845 NCGC00263039-01 1.0510 0.8580 0.9982 0.9481 0.9727 0.9645 NCGC00164631-03 0.7680 0.6690 0.3964 0.8420 1.2736 1.2682 NCGC00179302-02 1.1410 1.0030 0.9196 1.1533 0.9736 0.9618 NCGC00241113-01 0.9520 0.9790 1.0724 0.9410 1.3227 0.8627 NCGC00094381-04 8.1220 3.8710 1.1010 1.2849 1.4927 1.0845 NCGC00015546-04 1.0700 1.0230 1.1513 1.0553 1.1427 1.1555 NCGC00094381-03 18.6030 10.9800 1.5444 1.1125 1.2227 0.9227 NCGC00094381-05 18.0610 6.8950 2.8305 1.2069 0.8773 1.0045 NCGC00015545-02 0.4770 0.5490 0.5058 0.7586 0.9536 0.8627 NCGC00164631-04 0.6690 0.6490 0.3804 0.8803 0.9164 1.1336 NCGC00094112-04 0.5420 0.7190 0.7933 0.8123 0.8864 1.0409 NCGC00242500-01 7.0800 5.2430 3.3039 2.2432 1.9682 1.2482 NCGC00015545-07 0.5470 0.7010 0.8393 0.9434 1.1036 1.0736

TABLE 17 407 compounds chosen from the initial 1500 MLSMR “hit” compounds and the results from the Burkin lab rescreening in myoblasts (MB), myotubes (MT), and β-Gal stabilizing assay Both MB and β-Gal Less than 25% 407 compounds MB >25% MT >25% MT >25% Stabilizing or max increase in chosen for Burkin increase increase over increase over CMV both MB and Lab evaluation over DMSO DMSO DMSO activating MT NCGC repeatedly 52 (31%) 12 (7.2%)  10 (6%)    2* (1.2%) 90 (54%) active (166) Original top hits 6 (3%) 37 (18.8%) 12 (6.1%) 6** (3%)  136 (69%)  (197) SU9516 platform   6 (13.6%) 1 (2.3%)  3 (6.8%) 12*** (27.3%) 15 (34%) Analogs (44) *Neither of the β-Gal stabilizing compounds gave a >25% in α7^(+/LacZ) MB or MT assays **1 β-gal stabilizer also increased MT >25% over DMSO ***1 β-gal stabilizer also increased MB >25% over DMSO

Top Hits from the Screen

Next the top 6 compounds for activation of ITGA7 in myotubes, based on maximum response (Table 18) were selected and the screen was performed with a larger number of concentrations in order to achieve more accurate dose-response curves (FIG. 7). From these dose-response curves, GraphPad Prism nonlinear-regression analysis was used to determine the EC₅₀ in myotubes (Table 18). The maximum predicted increase was calculated as the maximum increase generated in the α7^(+/LacZ) multiplied by 2 due to the single allelic copy

TABLE 18 Summary of the top 6 compounds found to be effective in myotubes. Compound Known Online Online Screen EC50 MT FOLD Name MW effects Screen Score Fold-change α7^(+/LacZ) MT INCREASE SU9516 241.3 Cdk2 6.0 × 10⁻⁶ M 2.4 inhibitor MLS000532969 236.3 NA 44 1.7 2.2 × 10⁻⁶ M 2.0 MLS003126425 399.4 NA 43 1.9 7.0 × 10⁻⁶ M 2.0 MLS001060533 483.3 NA 42 1.9 4.7 × 10⁻⁶ M 1.5 MLS000683232 257.2 NA 5.3 × 10⁻⁶ M 1.7 MLS000683234 237.3 NA 46 2.6 2.7 × 10⁻⁶ M 1.7 SU9516 Increases α7 Integrin Protein in C2C12 Myotubes

In order to confirm the on-target activity of SU9516, C2C12 myotubes were treated with either DMSO control or 12 μM SU9516 for 48 hours. Western blot analysis of the myotube protein extracts were then performed for α7B Integrin and normalized to GAPDH (FIG. 8). SU9516 treated myotubes displayed an increase of approximately 1.6-fold in α7 Integrin protein levels compared to DMSO treated controls (FIG. 8). These results confirm the myogenic cell-based assay had successful identified novel small molecules that would target an increase in α7 integrin in skeletal muscle.

Duchenne muscular dystrophy is a fatal neuromuscular disease for which there is currently no cure and limited treatment options. Studies have shown that the α7β1 integrin is a major modifier of disease progression in mouse models of DMD. Loss of the α7 integrin in dystrophin deficient mdx mice results in more severe muscle disease, while transgenic or viral mediated expression of the α7 integrin can rescue mouse models of DMD. These results support the hypothesis that the α7β1 integrin is major modifier of disease progression in DMD. In support of the idea that drug-based modulation of α7β1 integrin in skeletal muscle may serve as a therapeutic avenue for DMD, it has been shown that prednisone, the current front line therapy for DMD, acts to increase laminin-α2 and α7β1 integrin in the muscles of mdx mice, GRMD dog and DMD patient cells. Together these preclinical studies indicate the α7 integrin is a “druggable” target for the treatment of DMD.

In this study a muscle cell-based assay was used to identify small molecules that promote an increase in α7 integrin in myogenic cells. A screen of FDA-approved and novel compound libraries identified several chemical platforms which may be promising for the treatment of DMD. From the FDA-approved libraries it was identified that the Fe-chelating compounds Ciclopirox and Deferoxamine as α7β1 integrin enhancers. In addition 2,2-dipyridyl which is within this same compound family was tested using the muscle cell-based assay and was confirmed to also increase α7 integrin levels. Since Ciclopirox, deferoxamine and 2,2-dipyridyl have been shown to increase stability of the transcription factor hypoxia inducible factor-1 (HIF-1) by preventing its breakdown, bioinformatic analysis was performed on the α7 integrin promoter. Analysis of a 2.8 kb fragment of the proximal α7 integrin promoter sequence using MATINSPECTOR (Genomatix) revealed the presence of a HIF-1 binding site and flanking sequences that promote HIF-1 binding. These results indicate Ciclopirox, Deferoxamine and 2,2-dipyridyl act to increase α7 integrin gene expression by inhibiting proteosomal breakdown of HIF-1 in muscle cells resulting in increased cellular levels of HIF-1 protein in skeletal muscle. Interestingly, increased HIF-1 levels are associated with increased angiogenesis and therefore not only would these drugs increase membrane stability through increased α7 integrin gene expression, but they may also increase muscle vascularization, improving blood flow and reducing the ischemia associated with dystrophic muscle.

To identify small novel molecular probes that increase the α7 integrin in skeletal muscle, the MLSMR at the National Chemical and Genomics Center (NCGC) at NIH was screened. Using the muscle cell-based assay 380,000 compounds in the Molecular Library and Small Molecule Repository (MLSMR) were screened using dose-response quantitative high through-put screening (qHTS). From the primary screen, 1,500 hit compounds were identified as actives. Analysis of these compounds using the hierarchical cluster approach revealed 321 clusters in which 17 clusters contained more than 10 compounds and 210 singletons. From these studies 500 compounds were cherry-picked for further testing which included confirmation of activity in the primary screening assay and secondary assays which included β-galactosidase reporter stabilizer, myostatin and cell-toxicity assays. In addition primary HTS active compounds were tested to determine if they increased β-galactosidase reporter in myoblasts and myotubes. From these counter-screens seven novel hit compounds were selected for further analysis to determine whether they increase the α7 integrin in a dose-dependent fashion. Analysis in mouse and DMD human muscle cells revealed that all seven increased both α7 integrin transcript and protein levels. These compounds gave a maximal increase in α7 integrin protein in human DMD muscle cells which would be in the therapeutic range as predicted from transgenic mouse studies.

Studies of the seven novel α7 integrin enhancing compounds showed that two molecular platforms are related, while the other compounds are unrelated to each other. One compound, SU9156 which was used as a positive control in the screen is currently in clinical trials as a potential anti-cancer therapeutic. SU9516 (3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one) is a 3-substituted-indolinone compound that binds to cdk2 and selectively inhibits its catalytic kinase activity. X-ray crystallography studies showed that the small molecule compound inhibited cdk2/cyclin A through competitive inhibition of ATP. In human leukemic cells, SU9516 caused the pronounced down-regulation of the anti-apoptotic protein Mcl-1 through transcriptional repression, increased proteasomal degradation, inhibition of RNA Pol II CTD phosphorylation and oxidative damage. Thus, SU9516 has demonstrated its potential as a viable pharmacological drug for the development of anti-neoplastic therapeutics and has reached clinical trials for the same. The molecular mechanism by which SU9516 enhances integrin is currently unknown. SU9516 has also been reported to inhibit glycogen synthase kinase 3β (GSK-3β), which is involved in normal cell death. Levels of inactive p-S9-GSK3β are reduced and total GSK3β is elevated in the muscles of patients with myotonic dystrophy type 1 (DM1). Inhibition of GSK3β in both DM1 cell culture and mouse models reduced muscle weakness and myotonia in DM1 mice. Hence, compounds normalizing GSK3β activity might be beneficial for improving muscle function in patients with DM1.

Using a novel muscle cell-based assay an exhaustive screen of small molecule compound libraries has been conducted and identified several FDA-approved and novel molecules that increase α7 integrin in skeletal muscle. These small molecules can serve as molecular probes to dissect the signaling pathways that regulate levels of the α7β1 integrin in skeletal muscle. In addition these molecules may serve as platforms to develop novel therapeutics that target an increase in α7 integrin for the treatment of muscular dystrophy.

Example 4

This example describes methods for identifying and analyzing suitable analog compounds useful for the methods and assays disclosed herein. In one embodiment, Stryka-969 was identified as a top “hit” from of the over 400,000 compounds screened using the assay disclosed herein due to its efficacy, large range of potency, and its lack of cellular toxicity (REF paper in prep). The chemical structure of Stryka-969 along with the dose-response curve generated in ITGA7^(+/LacZ) myotubes is illustrated FIG. 7. While Stryka-969 also functions equally well in myoblasts, the therapeutic target for enhanced α7 integrin protein are differentiated myofibers and thus this example focuses primarily on the Stryka-969 analogs function in ITGA7^(+/LacZ) myotubes. The maximum increase of ˜2-fold should translate into a 3-fold change in ITGA7 transcription due to the heterogenetic nature of the assay and single reporter. This increase is well within the therapeutic range previously reported for dystrophic mice.

FIG. 10 illustrates results obtained from some analogs of Stryka-969. These compounds were scored based on their structural similarity to Stryka-969 using the Tanimoto coefficient which measures the similarity between two. These compounds were then used to treat ITGA7^(+/LacZ) myotubes at eight different concentrations in order to produce dose-response curves (FIG. 10; n=3/concentration). The dose-response results suggest that the amide group may help increase α7 Integrin. This group is maintained in MLS000061149, which displays similar activity in the Burkin assay to the parent compound Stryka-969 (FIG. 10). The other compounds were less similar to Stryka-969 and displayed little to no relevant response (FIG. 10). Together, these studies indicate that SAR analysis will further elucidate the functional groups capable of maximizing the α7 integrin enhancing activity of this compound platform.

To assess the on-target effects of Stryka-969, cultured telomerase-expressing Human DMD myotubes were used. After a minimum of 10 days of differentiation, myotubes were treated with either DMSO or varying concentrations of Stryka-969. After 48 hours the media was aspirated and the myotubes were washed in PBS and then scraped into ice-cold RIPA buffer with protease inhibitors. After protein concentration was determined by BCA, standard SDS-PAGE and western blotting procedures for α7B Integrin and α-Tubulin were followed. A maximum increase of ˜1.7-fold over DMSO levels with a treatment of 20M Stryka-969 (FIG. 11) was obtained. This shows that Stryka-969 is an α7 integrin enhancing compound with strong therapeutic potential for the treatment of DMD patients.

Pre-clinical assessment of therapeutic small molecules, including ADMET studies, can help to prioritize small molecules with the highest therapeutic value. A step to ensuring that small molecules will be therapeutically on-target in patients is to use cultured Human patient cells for assessment. This ensures that given biologic availability has a conserved mechanism of action between species and the treatment should effectively produce the desired effect in the specific tissue.

Briefly, either 1 mg or 5 mg of each analog are suspended in DMSO at 10 μM concentration. Working plates are made to give a minimum of 8-point dose response per compound. Drugs are added to ITGA7^(+/LacZ) myotubes for 48 hours, at which time the FDG assay is performed as previously described. Using such analogs of Stryka-969 that achieve a minimum of 1.5-fold increase in the Burkin Lab ITGA7^(+/LacZ) based assay, 6 cm plates with cultured Human DMD myotubes are treated with a range of concentrations based on the dose-response discussed above (n=3/concentration). After 48 hours the myotubes are washed and lysed for western blot analysis. Several proteins, including α7B and β1A Integrins, Laminin-α2, and utrophin levels, are assessed using standard western blotting techniques. If compounds fail to produce a minimum of 1.5-fold α7 integrin increase they may be eliminated from further studies. The behavior of Stryka-969 on the cultured Human DMD myotubes discussed herein indicates that analogs of this compound may lead to increased α7 integrin in the Human cells. Western blots using a broad range of concentrations can be used to avoid missing an optimal dose for a small molecule, which could occur if the EC₁₀₀ concentration from the initial assay for use in the human DMD myotubes is solely relied upon.

The activity of other α7 integrin compounds also can be determined using β-galactosidase cleavage of the non-fluorescent compound fluorescein di-β-D-galactopyranoside (FDG) to fluorescein in both myoblasts and myotubes. For myoblast assays using analog compounds, 1×10⁴ cells are plated on 96 well black well culture plates and cells grown for 24 hours. The parent and analog compounds are added and the FDG bioassay is performed 48 hours after the addition of compounds. For myotube assays, 2.5×10⁵ myoblasts on 96 well black cell culture plates are grown for 24 hours. Cell differentiation medium is added daily and after 7 days of differentiation, and compounds are added to myotubes and incubated for 48 hours. β-galactosidase levels are then be quantified using the FDG assay. These assays re performed in triplicate for each compound screened. For each assay the parent compound and analogs are added to cells with a dose range from 0.5-20 μM in DMSO and a DMSO only control is added for each assay. The dose curve class and EC₅₀ for each compound is calculated, plotted and compared to the profile of the parent hit molecule. Analog molecules that show activity (both positive and negative) in myoblasts and myotubes inform the SAR for each molecular platform series. In parallel, microsome and plasma stability, permeability and solubility of confirmed lead compounds able to increase α7 integrin in myotubes is also evaluated to help determine the best templates for further pharmacokinetic evaluation. Lead compounds are selected for further studies based on the most potent activity in myotubes (the target tissue) and optimal physical and structural drug-like properties in accordance with Lipinski's Rules.

Positive analogs are then assessed to determine whether they increase α7 integrin transcript and protein using mouse C2C12 and human DMD myogenic cells. For myotube analysis, C2C12 and DMD myoblasts are differentiated in DMEM supplemented with 2% horse serum and 50 U/ml of penicillin/streptomycin. Compounds or DMSO are added to C2C12 and DMD myoblasts and myotubes at EC₁₀₀ dose calculated from the FDG screen above. All studies are done in triplicate, and RNA is extracted using TriZol reagent. RNA is reverse transcribed to cDNA using a Superscript II kit (Invitrogen). Primer sequences are used that specifically amplify mouse or human transcripts using SYBR Green technology and quantitative RT-PCR, and reactions carried out in an ABI Prism 7000 Sequence Detection System. The C_(T) value and a standard curve from a dilution series of cDNA from non-treated cells is calculated by the accompanying ABI Prism 7000 SDS software. Transcripts are normalized to 18S rRNA transcript and reported as fold change from control cells. Experiments are performed in triplicate and statistical significance (p<0.05) determined using ANOVA.

Immunoblot analysis is used to assess the protein level expression in treated and control cells. C2C12 and human DMD myoblasts and myotubes are cultured in triplicate experiments and treated with lead compounds or DMSO as described above. Cells are harvested using a cell scraper and extracted proteins. Extracted protein is quantified by a Bradford assay. Equal amounts of total protein are separated on SDS-PAGE gels at 40 mA for 1 hour and protein is transferred to nitrocellulose membranes. Blots are incubated with Ponceau S to confirm equal protein loading. Rabbit anti-α7 integrin antibodies, A2-345 and B2-347, at 1:500 with will be used to detect mouse and human α7A and α7B integrin respectively. Bands are scanned with an Odyssey Infrared Imaging System. Blots are re-probed with an anti-GAPDH to normalize band intensities for protein loading. A LiCoR Odyssey scanner and software is used to quantify band intensities and statistical significance (p<0.05) is determined using ANOVA.

The pharmacokinetic properties of the compounds having desirable drug-like structures are analyzed for in vivo activity. Three 8-week-old C57Bl/6 mice by will be treated by intraperiontoneal injection (i.p) with either the control solute or the compounds under investigation at the EC₁₀₀ determined in the examples discussed above. At 0 mins (before administration) and then 30 mins, 1 hr, 2 hrs, 4 hrs, 8 hrs, 12 hrs and 24 hrs after compound administration, 50 μl of blood is collected by retro-orbital bleeds and serum isolated by centrifugation. After administration, animals re observed and signs of toxicity including weight loss, convulsions, uncoordinated movement, torpidity, temperature changes, dispenia or death. In a separate series of pharmacokinetic experiments to assess muscle tissue levels of analogs, three 8-week-old C57Bl/6 mice are treated control solute or the compounds at the same time points listed above. At each time point mice are humanely euthanized by CO2 inhalation and the gastrocnemius, TA, heart, intestine and diaphragm muscles dissected for analysis. The muscle and intestine is extracted to determine the levels of each lead analog within the tissue.

Purified compounds serve as a control to identify the small molecule signature after LC-MS/MS analysis. Pharmacokinetic profiles include: serum half-life (t_(1/2)), Volume of distribution (V_(d)), drug concentration in serum (C_(o) or C_(SS)), Elimination rate constant (k_(e)), Clearance (CL), Bioavailability (f), peak serum concentration (C_(max)) and time to reach C_(max) (t_(max)). A lead compound with a suitable pharmacokinetic profile is then selected for further studies. Formulation is developed with the objective of developing a lead α7 integrin enhancing compound that can be orally administered. For experimental rigor, samples will be coded before analysis so those collecting data are blinded to the treatment group.

The pharmacokinetic profile for the lead compound identified above and the calculated optimal dose are used to determine if the on-target in vivo activity of the drug increases α7 integrin into the therapeutic range as determined by transgenic mouse studies. Off-target activity of the lead compound also will be examined. Three week-old mdx mice are administered with a suitable lead compound as determined using the above examples, or a vehicle by oral gavage daily for 14 days using dosing determined in pharmacokinetic studies. C57Bl/10 mice are included as wild-type controls. A minimum of 22 male mdx mice per experimental and control group are used as determined by Power analysis (Power=0.8, α=0.05 and r=0.5). Mice are weighed weekly and behavioral changes recorded. Mice are euthanized at 5 weeks-of-age and Tibialis anterior (TA), gastrocnemius, diaphragm and cardiac muscles harvested. Expression of α7 and β1 integrin is quantified by qRT-PCR and western blots as discussed herein. For off-target activity, transcript levels of integrin α3, α5, α6 and extracellular matrix genes are quantified. Primers are used that specifically amplify these mouse transcripts using SYBR Green technology and quantitative RT-PCR reactions performed in an ABI Prism 7000 Sequence Detection System. The C_(T) value for each is calculated using ABI Prism 7000 SDS software and transcripts normalized to 18S rRNA and reported as fold change from control tissue. Experiments are performed in triplicate and statistical significance (p<0.05) determined using ANOVA. Transcripts that change >2-fold are confirmed by western blot analysis.

To determine if the lead compound shows therapeutic benefit, a preliminary muscle histology study also is performed. Before harvesting tissue, mice are injected with Evans Blue dye (EBD) and histology performed for EBD uptake, percentage of myofibers with centrally located nuclei, inflammation, myofiber cross-sectional area and fibrosis as previously described. For experimental rigor, samples are coded before analysis so those collecting data will blinded to the treatment group. Data analyzed by ANOVA and a p-value<0.05 will be considered statistically significant.

These examples can be used to determine the PK/PD of a lead α7 integrin enhancing compound. The lead compound with favorable PK/PD is then assessed for on-target efficacy and off-target activity.

STATEMENTS OF THE DISCLOSURE

Paragraph 1. A method for treating a subject with muscular dystrophy, comprising administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one of or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy.

Paragraph 2. The method disclosed in the preceding paragraph, wherein the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1A (MDC1A), merosin deficient congenital muscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).

Paragraph 3. The method disclosed in any one of the preceding paragraphs wherein the muscular dystrophy is DMD, MDC1A or FCMD.

Paragraph 4. The method disclosed in any one of the preceding paragraphs, wherein the muscular dystrophy is DMD.

Paragraph 5. The method disclosed in any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.

Paragraph 6. The method disclosed in the preceding paragraph, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.

Paragraph 7. The method disclosed in paragraph 6, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog, a different compound selected from Formulas 1-16 and/or Tables 1-16 and 18.

Paragraph 8. The method disclosed in any one of the preceding paragraphs, further comprising selecting a subject with muscular dystrophy.

Paragraph 9. The method disclosed in the preceding paragraphs, wherein selecting a subject with muscular dystrophy comprises diagnosing the subject with muscular dystrophy prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.

Paragraph 10. A method of enhancing muscle regeneration, repair, or maintenance in a subject, comprising:

administering an effective amount of an α7β1 integrin modulatory agent to the subject in need of muscle regeneration, repair, or maintenance, wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one of or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby enhancing muscle regeneration, repair or maintenance in a subject.

Paragraph 11. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein the α7β1 modulatory agent is administered prior to the subject experiencing muscle damage or disease.

Paragraph 12. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the method is a method of enhancing muscle maintenance in a subject.

Paragraph 13. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered to the subject prior to the subject exercising.

Paragraph 14. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered to a subject at risk of acquiring a muscle disease or damage.

Paragraph 15. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, further comprising selecting a subject in need of enhancing muscle regeneration, repair, or maintenance.

Paragraph 16. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired muscle regeneration prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.

Paragraph 17. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein selecting a subject in need of enhancing muscle regeneration, repair, or maintenance comprises diagnosing the subject with a condition characterized by impaired production of a component of α7β1 integrin prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject.

Paragraph 18. The method of enhancing muscle regeneration, repair, or maintenance in a subject of any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.

Paragraph 19. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.

Paragraph 20. The method of enhancing muscle regeneration, repair, or maintenance in a subject of the preceding paragraph, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog, or a different compound selected from Formulas 1-16 and/or Tables 1-16 and 18.

Paragraph 21. A method of prospectively preventing or reducing muscle injury or damage in a subject, comprising, administering an effective amount of an α7β1 integrin modulatory agent to the subject wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby prospectively preventing or reducing muscle injury or damage in the subject.

Paragraph 22. The method of the preceding paragraph, wherein the subject is at risk of developing a muscle injury or damage.

Paragraph 23. The method of prospectively preventing or reducing muscle injury or damage in a subject in any one of the preceding paragraphs, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.

Paragraph 24. The method of prospectively preventing or reducing muscle injury or damage in a subject of the preceding paragraph, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.

Paragraph 25. The method of prospectively preventing or reducing muscle injury or damage in a subject of the preceding paragraph, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog, or a different compound selected from Formulas 1-16 and/or Tables 1-16 and 18.

Paragraph 26. A method of enhancing α7β1 integrin expression, comprising contacting a cell with an effective amount of an α7β1 integrin modulatory agent, wherein the α7β1 integrin modulatory agent is selected from any one or more of Formulas 1-16 and/or any one or more of the compounds disclosed in Tables 1-16 and 18 and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression in the treated cell relative to α7β1 integrin expression in an untreated cell, thereby enhancing α7β1 integrin expression.

Paragraph 27. The method of the preceding paragraph, wherein the cell is a muscle cell.

Paragraph 28. The method of enhancing α7β1 integrin expression of any of the preceding paragraphs, wherein the muscle cell is present in a mammal, and wherein contacting the cell with an agent comprises administering the agent to the mammal.

In view of the many possible embodiments to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated embodiments are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims. 

We claim:
 1. A method for treating a subject with muscular dystrophy, comprising administering an effective amount of an α7β1 integrin modulatory agent to the subject with muscular dystrophy, wherein the α7β1 integrin modulatory agent is

and wherein the α7β1 integrin modulatory agent increases α7β1 integrin expression or activity as compared to α7β1 integrin expression or activity prior to treatment, thereby treating the subject with muscular dystrophy.
 2. The method of claim 1, wherein the muscular dystrophy is merosin deficient congenital muscular dystrophy Type 1A (MDC1A), merosin deficient congenital muscular dystrophy Type 1D (MDC1D), limb-girdle muscular dystrophy (LGMD), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD) or Facioscapulohumeral muscular dystrophy (FHMD).
 3. The method of claim 2, wherein the muscular dystrophy is DMD, MDC1A or FCMD.
 4. The method of claim 2, wherein the muscular dystrophy is DMD.
 5. The method of claim 1, wherein the α7β1 integrin modulatory agent is administered with an additional therapeutic agent.
 6. The method of claim 5, wherein the additional therapeutic agent is a costameric protein, a growth factor, satellite cells, stem cells, myocytes or an additional α7β1 integrin modulatory agent.
 7. The method of claim 6, wherein the additional α7β1 integrin modulatory agent is laminin-111, a laminin-111 fragment, valproic acid, or a valproic acid analog.
 8. The method of claim 1, further comprising selecting a subject with muscular dystrophy.
 9. The method of claim 8, wherein selecting a subject with muscular dystrophy comprises diagnosing the subject with muscular dystrophy prior to administering an effective amount of the α7β1 integrin modulatory agent to the subject. 